1 00:00:00,500 --> 00:00:03,270 The following content is provided under a Creative 2 00:00:03,270 --> 00:00:04,630 Commons license. 3 00:00:04,630 --> 00:00:07,140 Your support will help MIT OpenCourseWare 4 00:00:07,140 --> 00:00:11,470 continue to offer high quality educational resources for free. 5 00:00:11,470 --> 00:00:14,100 To make a donation or view additional materials 6 00:00:14,100 --> 00:00:18,050 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:18,050 --> 00:00:19,000 at ocw.mit.edu. 8 00:00:27,040 --> 00:00:31,060 JOANNE STUBBE: This recitation on mass spec 9 00:00:31,060 --> 00:00:35,480 is supposedly associated with reactive oxygen species. 10 00:00:35,480 --> 00:00:39,400 So [INAUDIBLE],, which happens all the time in this course, 11 00:00:39,400 --> 00:00:44,340 because we can't describe all the techniques as we go along. 12 00:00:44,340 --> 00:00:46,210 So what I'm going to do is just give you 13 00:00:46,210 --> 00:00:49,570 a two second overview of what you 14 00:00:49,570 --> 00:00:53,020 need to think about to put the paper you 15 00:00:53,020 --> 00:00:54,520 read into the big picture. 16 00:00:54,520 --> 00:00:55,830 I don't think the paper-- 17 00:00:55,830 --> 00:00:58,060 the paper also explains it. 18 00:00:58,060 --> 00:01:01,060 And this week we're going to focus 19 00:01:01,060 --> 00:01:05,110 on the mass spec paper, which is mostly 20 00:01:05,110 --> 00:01:07,940 sort of trying to figure out the technology, 21 00:01:07,940 --> 00:01:12,160 and then the next week is focused on the biology. 22 00:01:12,160 --> 00:01:16,550 And so the major unsolved problem-- 23 00:01:16,550 --> 00:01:21,400 so everybody and his brother is using mass spectrometry 24 00:01:21,400 --> 00:01:23,384 as a tool nowadays. 25 00:01:23,384 --> 00:01:28,840 There has been a revolution in mass spectrometry. 26 00:01:28,840 --> 00:01:34,300 [INAUDIBLE] 27 00:01:34,300 --> 00:01:37,540 The instrumentation is cheaper. 28 00:01:37,540 --> 00:01:43,360 The mass spectrometric methods have just really taken off. 29 00:01:43,360 --> 00:01:47,590 And people didn't even know who mass spectrometrists were, 30 00:01:47,590 --> 00:01:50,080 but they're starting to win major prizes, because it's 31 00:01:50,080 --> 00:01:53,470 revolutionized what we can do. 32 00:01:53,470 --> 00:01:56,710 I was talking to somebody yesterday, 33 00:01:56,710 --> 00:02:02,360 and they just got a mass on a protein that's 3.3 million. 34 00:02:02,360 --> 00:02:04,030 How do you get a protein into the gas 35 00:02:04,030 --> 00:02:06,020 phase that's 3.3 million? 36 00:02:06,020 --> 00:02:07,270 Right. 37 00:02:07,270 --> 00:02:10,070 Doesn't that sort of blow your mind? 38 00:02:10,070 --> 00:02:13,400 Anyhow, it's been a revolution. 39 00:02:13,400 --> 00:02:17,980 And we're going to be looking at-- this module seven, which 40 00:02:17,980 --> 00:02:22,240 is on reactive oxygen species, and we've 41 00:02:22,240 --> 00:02:25,930 been talking about the question of homeostasis. 42 00:02:25,930 --> 00:02:30,320 And so one of the things with these reactive oxygen species 43 00:02:30,320 --> 00:02:36,420 is they are used by us to kill bacteria, viruses, 44 00:02:36,420 --> 00:02:37,670 or parasites. 45 00:02:37,670 --> 00:02:41,510 But now, in the last five years or so, everybody's 46 00:02:41,510 --> 00:02:44,420 focusing on the fact that here are these reactive oxygen 47 00:02:44,420 --> 00:02:50,600 species that play a key role in signaling, which is everywhere, 48 00:02:50,600 --> 00:02:53,410 and the signaling process we're going 49 00:02:53,410 --> 00:03:01,070 to be looking at next time and is alluded to 50 00:03:01,070 --> 00:03:05,540 in this particular paper is epidermal growth factor 51 00:03:05,540 --> 00:03:08,270 receptor and epidermal growth factor. 52 00:03:08,270 --> 00:03:11,210 There are hundreds of these proteins that 53 00:03:11,210 --> 00:03:14,210 have receptors that are involved in growth 54 00:03:14,210 --> 00:03:18,190 and epidermal growth factor receptor [INAUDIBLE] 55 00:03:18,190 --> 00:03:20,950 of successful cancer therapeutics. 56 00:03:20,950 --> 00:03:23,650 So it's interesting what happens up here, 57 00:03:23,650 --> 00:03:27,130 what happens down here, how do you control all of that, 58 00:03:27,130 --> 00:03:28,700 and people are studying this. 59 00:03:28,700 --> 00:03:33,350 So we've already seen cystine is unique. 60 00:03:33,350 --> 00:03:35,690 And if you have a reactive oxygen species, 61 00:03:35,690 --> 00:03:39,115 and we'll see that the reactive oxygen species 62 00:03:39,115 --> 00:03:41,740 we'll be looking at, when we're going to be looking at a number 63 00:03:41,740 --> 00:03:43,240 is actually superoxide. 64 00:03:43,240 --> 00:03:46,480 So that's one electron reduced oxygen, 65 00:03:46,480 --> 00:03:49,470 which in the presence of protons can rapidly disproportionally 66 00:03:49,470 --> 00:03:52,780 give[?] oxygen gas to hydrogen peroxide. 67 00:03:52,780 --> 00:04:02,060 And hydrogen peroxide can react with cystines 68 00:04:02,060 --> 00:04:08,000 to form sulfenic acids, which is the subject of the paper you 69 00:04:08,000 --> 00:04:08,810 had to read. 70 00:04:11,590 --> 00:04:15,620 And so the question is how prevalent is this, 71 00:04:15,620 --> 00:04:19,700 and the question is, is this important and interesting 72 00:04:19,700 --> 00:04:23,330 in terms of regulation inside the cell? 73 00:04:23,330 --> 00:04:26,010 And so the key issue is-- 74 00:04:26,010 --> 00:04:28,640 even cystines aren't all that stable, you know, 75 00:04:28,640 --> 00:04:30,562 if you have proteins with cystines, 76 00:04:30,562 --> 00:04:33,020 and you let it sit around for a long time, you could form-- 77 00:04:33,020 --> 00:04:35,600 and the protein's concentrated, you could form disulfides. 78 00:04:35,600 --> 00:04:37,220 It's not a straightforward reaction, 79 00:04:37,220 --> 00:04:40,531 but you can form disulfides. 80 00:04:40,531 --> 00:04:44,060 The question is if you had hydrogen peroxide 81 00:04:44,060 --> 00:04:47,410 inside the cell, which you do, can you form sulfenic acids, 82 00:04:47,410 --> 00:04:51,660 and do they have a consequence biologically? 83 00:04:51,660 --> 00:04:53,610 OK, and that's the question we're 84 00:04:53,610 --> 00:04:55,210 going to address next time. 85 00:04:55,210 --> 00:04:56,955 And so the issue is this is unstable. 86 00:05:00,370 --> 00:05:03,090 So if you want to develop a method 87 00:05:03,090 --> 00:05:07,400 to look for this species, and you start cracking open cells, 88 00:05:07,400 --> 00:05:09,870 and you start working it up, what happens 89 00:05:09,870 --> 00:05:12,430 is this falls apart and reacts and gets destroyed. 90 00:05:12,430 --> 00:05:17,950 And an example of this is the area of DNA therapeutics 91 00:05:17,950 --> 00:05:20,630 and DNA drug interactions, therapeutics that interact 92 00:05:20,630 --> 00:05:21,130 with DNA. 93 00:05:21,130 --> 00:05:24,610 For decades, you see lesions on your DNA. 94 00:05:24,610 --> 00:05:26,360 How do you determine what the lesions are? 95 00:05:26,360 --> 00:05:29,740 Mass spec has been a major method to look at that. 96 00:05:29,740 --> 00:05:32,990 Almost all the lesions in the early days 97 00:05:32,990 --> 00:05:36,160 were complete artifacts of the analytical chemistry 98 00:05:36,160 --> 00:05:37,530 to work them up. 99 00:05:37,530 --> 00:05:39,160 They had to get them into some form 100 00:05:39,160 --> 00:05:42,520 that you could stabilize the lesion and then analyze it. 101 00:05:42,520 --> 00:05:44,810 And what was happening because they weren't 102 00:05:44,810 --> 00:05:47,400 careful enough and quantitative enough, 103 00:05:47,400 --> 00:05:50,250 they changed it to something else. 104 00:05:50,250 --> 00:05:52,460 And so the data in the early years 105 00:05:52,460 --> 00:05:54,630 was all completely misinterpreted. 106 00:05:54,630 --> 00:05:58,260 So the issue in this paper is that other people 107 00:05:58,260 --> 00:06:02,390 had developed this, and Kate Caroll has taken this on. 108 00:06:02,390 --> 00:06:04,880 Can we have a way of derivativizing 109 00:06:04,880 --> 00:06:10,242 this [? mentally ?] inside the cell, because if you disrupt-- 110 00:06:10,242 --> 00:06:14,450 if you disrupt this by cracking open the cells 111 00:06:14,450 --> 00:06:17,810 and trying to purify things, it undergoes further reaction, 112 00:06:17,810 --> 00:06:20,660 and what this can undergo further reaction to 113 00:06:20,660 --> 00:06:25,700 is SO2 minus, sulfonic acids or SO3 minus. 114 00:06:28,660 --> 00:06:31,080 Sulfinic acids and sulfonic acids. 115 00:06:31,080 --> 00:06:31,760 OK. 116 00:06:31,760 --> 00:06:37,010 And it turns out this reaction is also reversible 117 00:06:37,010 --> 00:06:38,950 with hydrogen peroxide a lot of people 118 00:06:38,950 --> 00:06:43,320 are looking at that at this stage is irreversible. 119 00:06:43,320 --> 00:06:43,950 Anyhow. 120 00:06:43,950 --> 00:06:46,079 So the question is can you develop methods 121 00:06:46,079 --> 00:06:47,370 to look at all of these things. 122 00:06:47,370 --> 00:06:50,330 And in fact Tannenbaum, who was in the chemistry department, 123 00:06:50,330 --> 00:06:55,840 but also [INAUDIBLE],, he is looking at nitrosation of SHs, 124 00:06:55,840 --> 00:06:58,190 again forming a reactive species, 125 00:06:58,190 --> 00:06:59,890 and he's developed new methods sort of 126 00:06:59,890 --> 00:07:02,730 like Carroll has to try to specifically look 127 00:07:02,730 --> 00:07:03,860 at these modifications. 128 00:07:03,860 --> 00:07:06,672 And in the end, what you want to do, and this is the key, 129 00:07:06,672 --> 00:07:09,130 you might be able to detect this-- the question is, is this 130 00:07:09,130 --> 00:07:10,600 interesting? 131 00:07:10,600 --> 00:07:12,700 So you have to have a way to connect this back 132 00:07:12,700 --> 00:07:15,170 to the biology inside the cell. 133 00:07:15,170 --> 00:07:18,220 And that's what the second paper is focused on. 134 00:07:18,220 --> 00:07:20,740 So what we're doing today is simply 135 00:07:20,740 --> 00:07:22,990 looking at the technology that's been developed 136 00:07:22,990 --> 00:07:25,300 to try to get a handle, how do you 137 00:07:25,300 --> 00:07:28,750 look at sulfenylation, you're not really 138 00:07:28,750 --> 00:07:31,940 focusing on the biology of the consequences. 139 00:07:31,940 --> 00:07:35,400 And so what we're using is mass spec. 140 00:07:35,400 --> 00:07:37,750 And we're using a method of mass-- 141 00:07:37,750 --> 00:07:41,220 how many of you have done mass spec? 142 00:07:41,220 --> 00:07:45,100 So if you know something and I say something wrong, 143 00:07:45,100 --> 00:07:48,700 you should speak up, because I'm not a mass spec expert. 144 00:07:48,700 --> 00:07:52,650 And actually, I've got a whole bunch of information 145 00:07:52,650 --> 00:07:55,840 from, say, the Broad, and I thought it was not very good. 146 00:07:55,840 --> 00:07:58,705 So we need a way of trying to figure out 147 00:07:58,705 --> 00:08:01,080 that you're going to see-- there's hundreds of variations 148 00:08:01,080 --> 00:08:01,621 on the theme. 149 00:08:01,621 --> 00:08:04,160 I'm going to give you a very simplified overview of what 150 00:08:04,160 --> 00:08:06,590 things you need to think about. 151 00:08:06,590 --> 00:08:12,530 And so if I say something that you don't agree with, tell me. 152 00:08:12,530 --> 00:08:15,420 OK, so when looking at mass spec-- 153 00:08:15,420 --> 00:08:17,820 this didn't exist when I was your age-- 154 00:08:17,820 --> 00:08:22,060 using soft ionization methods, and what does that mean? 155 00:08:22,060 --> 00:08:27,090 It means that you don't want your molecules to crack. 156 00:08:27,090 --> 00:08:30,120 So the issue is that what mass spec 157 00:08:30,120 --> 00:08:32,080 is about-- so really looking at mass spec, 158 00:08:32,080 --> 00:08:35,190 and the key issue of what you wind up looking at 159 00:08:35,190 --> 00:08:38,580 is mass to charge. 160 00:08:41,299 --> 00:08:42,165 OK. 161 00:08:42,165 --> 00:08:43,440 So m over z. 162 00:08:43,440 --> 00:08:48,800 OK, so the problem is how do we get something charged 163 00:08:48,800 --> 00:08:54,450 enough so that the mass is small enough so that you can see it, 164 00:08:54,450 --> 00:08:57,320 taking a look at the mass analyzer, which is going to be 165 00:08:57,320 --> 00:08:59,720 part of all mass spectrometers. 166 00:08:59,720 --> 00:09:00,320 OK. 167 00:09:00,320 --> 00:09:04,697 So there are two different ways you could 168 00:09:04,697 --> 00:09:05,780 change the mass to charge. 169 00:09:05,780 --> 00:09:07,780 You could dump an electron in. 170 00:09:07,780 --> 00:09:09,950 And if you dump an electron in, that 171 00:09:09,950 --> 00:09:13,690 produces radical species, which can then fragment. 172 00:09:13,690 --> 00:09:14,910 We want to avoid that. 173 00:09:14,910 --> 00:09:19,200 That's not soft ionization methods. 174 00:09:19,200 --> 00:09:21,260 But how can we control this? 175 00:09:21,260 --> 00:09:26,870 The way we can control this is dumping in protons. 176 00:09:26,870 --> 00:09:33,370 So what we do is we can control it by adding protons 177 00:09:33,370 --> 00:09:36,050 or by subtracting protons. 178 00:09:40,400 --> 00:09:43,382 And we'll see that the different methods 179 00:09:43,382 --> 00:09:44,840 we're going to be looking at, we'll 180 00:09:44,840 --> 00:09:46,970 see there are two main methods that most of you 181 00:09:46,970 --> 00:09:49,670 have probably heard about your classes. 182 00:09:49,670 --> 00:09:55,508 One is electrode spray ionization, so ESI. 183 00:09:55,508 --> 00:09:59,150 And I think, if you're in Brad's lab, they have a lot of these. 184 00:10:04,010 --> 00:10:08,846 Yesterday's class had people that had used these, 185 00:10:08,846 --> 00:10:10,220 but really didn't know much about 186 00:10:10,220 --> 00:10:11,340 what's inside the machine. 187 00:10:11,340 --> 00:10:12,920 So this is the kind of thing I think 188 00:10:12,920 --> 00:10:15,280 your generation, if you're going to use this as a tool, 189 00:10:15,280 --> 00:10:18,230 need to roll up your sleeves and understand 190 00:10:18,230 --> 00:10:29,010 a lot more about what's going on, and MALDI, maser MALDI. 191 00:10:29,010 --> 00:10:32,030 Matrix Assisted Laser Desorption-- 192 00:10:32,030 --> 00:10:35,190 it will become clear why it's called that in a minute. 193 00:10:41,160 --> 00:10:42,610 So these are the two methods. 194 00:10:42,610 --> 00:10:47,620 And what we do is we can protonate, 195 00:10:47,620 --> 00:10:52,030 so that we can move this into the analyzer range, where 196 00:10:52,030 --> 00:10:53,120 we can actually read it. 197 00:10:53,120 --> 00:10:55,330 So what we'll see is the analyzer-- 198 00:10:55,330 --> 00:10:57,307 I'm going to show you sort of what 199 00:10:57,307 --> 00:10:59,140 the three parts of a mass spectrometer are-- 200 00:10:59,140 --> 00:11:03,210 can only read 1,000 to 2,000 daltons. 201 00:11:03,210 --> 00:11:06,190 OK, so if you look at your protein, much, much bigger. 202 00:11:06,190 --> 00:11:09,610 So you're going to have to stick a lot of charges on there 203 00:11:09,610 --> 00:11:11,150 to be able to see anything. 204 00:11:11,150 --> 00:11:13,880 So that's the whole thing, and the question is, 205 00:11:13,880 --> 00:11:18,690 how do you do it by one method or by using the other method? 206 00:11:18,690 --> 00:11:26,644 OK, so all mass specs have sort of the same components. 207 00:11:30,600 --> 00:11:36,730 And you can go to websites. 208 00:11:36,730 --> 00:11:39,470 The Broad does have a website, and what the Broad will 209 00:11:39,470 --> 00:11:42,500 tell you is what all these spectrometers are, 210 00:11:42,500 --> 00:11:44,930 but I don't think they do a particularly good job telling 211 00:11:44,930 --> 00:11:47,956 you what's useful for what, and why it's useful, 212 00:11:47,956 --> 00:11:49,580 which is, I think, what you need to use 213 00:11:49,580 --> 00:11:53,660 if you're only going to use it fleetingly and then move out. 214 00:11:53,660 --> 00:11:55,410 So you have a source. 215 00:11:55,410 --> 00:11:56,540 So you have an inlet. 216 00:11:56,540 --> 00:12:00,410 How do you get your sample from the liquid phase 217 00:12:00,410 --> 00:12:02,760 or the solid phase into the gas phase? 218 00:12:02,760 --> 00:12:04,900 OK, so that's going to be that. 219 00:12:04,900 --> 00:12:10,290 And so what is the distinct ionization method? 220 00:12:13,650 --> 00:12:18,140 And we will see that there are many ways that you can ionize, 221 00:12:18,140 --> 00:12:21,290 and we're just going to briefly look at in a cartoon 222 00:12:21,290 --> 00:12:23,930 overview of how this happens. 223 00:12:23,930 --> 00:12:26,900 And then so once you ionize it, it 224 00:12:26,900 --> 00:12:30,770 needs to move from the source. 225 00:12:30,770 --> 00:12:38,910 So you need to have ion movement into the analyzer. 226 00:12:38,910 --> 00:12:44,540 So this is the mass analyzer. 227 00:12:44,540 --> 00:12:46,540 And this becomes important. 228 00:12:46,540 --> 00:12:53,720 And we will see in a second that there are many methods 229 00:12:53,720 --> 00:12:58,440 to do the mass analysis, mass to charge analysis, 230 00:12:58,440 --> 00:13:03,840 and then after you do this, you have a detector. 231 00:13:03,840 --> 00:13:06,860 And then, furthermore-- and I think this is a big part of it 232 00:13:06,860 --> 00:13:10,834 now, if you're doing wholesale anything, 233 00:13:10,834 --> 00:13:12,750 you have to have a really sophisticated method 234 00:13:12,750 --> 00:13:14,690 of data analysis. 235 00:13:14,690 --> 00:13:19,260 And so that's the other thing that I get frustrated 236 00:13:19,260 --> 00:13:21,160 about all the time. 237 00:13:21,160 --> 00:13:22,620 So you see people-- 238 00:13:22,620 --> 00:13:25,890 I mean, people do experiments where they've spent-- 239 00:13:25,890 --> 00:13:28,050 last year, somebody spent three months 240 00:13:28,050 --> 00:13:30,450 trying to get all the proteins out of a cell, 241 00:13:30,450 --> 00:13:33,900 10,000 proteins out of the cell by mass spectrometry right. 242 00:13:33,900 --> 00:13:36,000 Now, because the technology is changing, 243 00:13:36,000 --> 00:13:37,920 they can do it in four days. 244 00:13:37,920 --> 00:13:39,870 But what do you do with all this information? 245 00:13:39,870 --> 00:13:42,404 And how do you use this information 246 00:13:42,404 --> 00:13:44,820 in a constructive way, and how do you know if it's correct 247 00:13:44,820 --> 00:13:45,340 or not? 248 00:13:45,340 --> 00:13:46,769 So those are the kinds of things. 249 00:13:46,769 --> 00:13:48,310 I think if you're going to use this-- 250 00:13:48,310 --> 00:13:50,840 I think everybody is going to be using this technology. 251 00:13:50,840 --> 00:13:55,740 You need to educate yourself about how to look at this. 252 00:13:55,740 --> 00:13:57,670 OK, so that's what the issue is. 253 00:13:57,670 --> 00:14:05,280 And so we have a source, an analyzer, and a detector. 254 00:14:05,280 --> 00:14:09,030 OK, so this is just a cartoon of that, which 255 00:14:09,030 --> 00:14:11,170 describes this in more detail. 256 00:14:11,170 --> 00:14:13,320 And I think he put this on the web. 257 00:14:13,320 --> 00:14:15,270 I think he put the PowerPoint on the web. 258 00:14:15,270 --> 00:14:17,910 I was doing this at the last minute yesterday. 259 00:14:17,910 --> 00:14:19,780 So it's different from the handout 260 00:14:19,780 --> 00:14:21,030 I gave you that's written out. 261 00:14:21,030 --> 00:14:22,110 This is a PowerPoint. 262 00:14:22,110 --> 00:14:24,390 OK, so you can go back and look at this, 263 00:14:24,390 --> 00:14:27,090 but one of the other things I wanted to say 264 00:14:27,090 --> 00:14:31,380 is that sometimes when you analyze your mass, 265 00:14:31,380 --> 00:14:36,089 you want to analyze it further, and that was true-- 266 00:14:36,089 --> 00:14:37,630 many of you might not have caught it, 267 00:14:37,630 --> 00:14:39,640 but that was true in the analysis that 268 00:14:39,640 --> 00:14:41,980 was carried out in this paper. 269 00:14:41,980 --> 00:14:43,605 Did anybody recognize that you had 270 00:14:43,605 --> 00:14:48,598 to analyze this using more than one mass spec? 271 00:14:48,598 --> 00:14:50,650 Did you look at the data carefully enough? 272 00:14:57,202 --> 00:15:00,380 So also you probably didn't read the supplementary information, 273 00:15:00,380 --> 00:15:03,560 which also is critical to think about. 274 00:15:03,560 --> 00:15:05,480 I mean, if you want to look at the methods, 275 00:15:05,480 --> 00:15:08,000 you need to get in there and roll up your sleeves 276 00:15:08,000 --> 00:15:08,750 and look at them. 277 00:15:08,750 --> 00:15:12,620 So we're going to see that the methods that people often use 278 00:15:12,620 --> 00:15:14,630 is they don't look at the whole protein, 279 00:15:14,630 --> 00:15:17,410 but they degrade it down into pieces. 280 00:15:17,410 --> 00:15:22,310 So then you can find here a whole bunch of pieces, OK. 281 00:15:22,310 --> 00:15:26,320 But that doesn't tell you anything. 282 00:15:26,320 --> 00:15:28,010 The mass does tell you something. 283 00:15:28,010 --> 00:15:30,260 It might tell you whether it's sulfenylated 284 00:15:30,260 --> 00:15:32,000 or hopefully, you can distinguish 285 00:15:32,000 --> 00:15:34,045 between any other modification, but it 286 00:15:34,045 --> 00:15:37,430 doesn't tell you the location of the sulfenylation. 287 00:15:37,430 --> 00:15:40,640 And so you can do a second method. 288 00:15:40,640 --> 00:15:43,520 So you could have some other gas. 289 00:15:43,520 --> 00:15:46,310 There are many ways to do this that you bring this in 290 00:15:46,310 --> 00:15:47,950 to now take a peptide. 291 00:15:47,950 --> 00:15:50,180 So you pick one mass charge. 292 00:15:50,180 --> 00:15:53,450 You throw in something that's going to degrade it 293 00:15:53,450 --> 00:15:56,030 by fragmentation, and then I'll show you 294 00:15:56,030 --> 00:15:58,700 in a minute we understand what kind of-- 295 00:15:58,700 --> 00:16:01,670 using certain methods, we understand the fragmentation 296 00:16:01,670 --> 00:16:03,170 patterns, which actually allow you 297 00:16:03,170 --> 00:16:05,210 to sequence the amino acids. 298 00:16:05,210 --> 00:16:08,420 And the reason I'm bringing that in is when I first got to MIT, 299 00:16:08,420 --> 00:16:11,720 Klaus Biemann was in the lab, and I did many experiments 300 00:16:11,720 --> 00:16:13,204 with him. 301 00:16:13,204 --> 00:16:14,870 And these are the first experiments that 302 00:16:14,870 --> 00:16:18,830 were done to sequence peptides by mass spec as 303 00:16:18,830 --> 00:16:21,540 opposed to doing Edman sequencing, 304 00:16:21,540 --> 00:16:24,080 which the mass spec was actually better, 305 00:16:24,080 --> 00:16:25,730 and there are pluses and minuses, 306 00:16:25,730 --> 00:16:27,950 but I noticed from looking at the literature, 307 00:16:27,950 --> 00:16:31,610 people were still using the same method that he developed. 308 00:16:35,330 --> 00:16:37,860 So this is just a cartoon. 309 00:16:37,860 --> 00:16:42,260 And it just shows you that there are many ionization methods. 310 00:16:42,260 --> 00:16:47,870 We're focusing on these two, FAB, fast atom bombardment. 311 00:16:47,870 --> 00:16:50,320 We didn't have any of these when I was your age. 312 00:16:50,320 --> 00:16:52,880 Fast atom bombardment was something 313 00:16:52,880 --> 00:16:57,140 I used a lot because I've worked on DNA drug interactions, 314 00:16:57,140 --> 00:17:00,060 and it allows you to look at nucleic acids. 315 00:17:00,060 --> 00:17:01,730 And a lot of these other methods don't. 316 00:17:01,730 --> 00:17:03,620 I mean, we're focused on proteomics 317 00:17:03,620 --> 00:17:08,450 in this particular paper, and then mass analyzer. 318 00:17:08,450 --> 00:17:09,829 So you have time of flight. 319 00:17:09,829 --> 00:17:13,349 I think Brad's lab has MALDI time of flight. 320 00:17:13,349 --> 00:17:14,510 So what does that mean? 321 00:17:14,510 --> 00:17:18,680 You've got a long tube in here, and what happens 322 00:17:18,680 --> 00:17:21,250 is you have mass to charge, and they're different sizes. 323 00:17:21,250 --> 00:17:23,390 And so the smaller ones fly faster. 324 00:17:23,390 --> 00:17:25,369 They don't want to keep away from the walls, 325 00:17:25,369 --> 00:17:28,430 but the smaller ones fly faster than the bigger ones. 326 00:17:28,430 --> 00:17:32,030 So that helps you differentiate between all the ions 327 00:17:32,030 --> 00:17:34,510 you're actually looking at. 328 00:17:34,510 --> 00:17:36,860 I guess somebody just told me you guys just 329 00:17:36,860 --> 00:17:40,680 got a new quadrupole ion trap. 330 00:17:40,680 --> 00:17:42,300 Anyhow, if you want to look at this, 331 00:17:42,300 --> 00:17:44,050 I have notes on all these things. 332 00:17:44,050 --> 00:17:47,780 But I think this is something you'd have to study in detail. 333 00:17:47,780 --> 00:17:49,290 And so while I have pictures of them 334 00:17:49,290 --> 00:17:51,360 all and how you can differentiate one 335 00:17:51,360 --> 00:17:53,069 from the other, I think it doesn't really 336 00:17:53,069 --> 00:17:55,109 mean that much to me, because I don't know enough 337 00:17:55,109 --> 00:17:57,090 about the physics of how they were designed. 338 00:17:57,090 --> 00:18:01,860 I mean, this really has revolutionized what you can do. 339 00:18:01,860 --> 00:18:02,360 OK. 340 00:18:02,360 --> 00:18:05,970 So that's the components of all mass spectrometry. 341 00:18:05,970 --> 00:18:08,580 What I want to do is very just briefly look at the ESI 342 00:18:08,580 --> 00:18:11,250 and then look at the MALDI and then 343 00:18:11,250 --> 00:18:13,110 show you what the issues are in general, 344 00:18:13,110 --> 00:18:16,200 and then we'll focus right in on the paper, 345 00:18:16,200 --> 00:18:19,170 and the recitation I did on Thursday, 346 00:18:19,170 --> 00:18:21,030 we didn't quite get through all of it. 347 00:18:21,030 --> 00:18:24,090 We got through most of it, but then we'll continue next week 348 00:18:24,090 --> 00:18:26,670 and also attach this to the biology, which 349 00:18:26,670 --> 00:18:30,900 is the second paper, the nature chemical biology paper also 350 00:18:30,900 --> 00:18:33,390 written by the Carroll group. 351 00:18:37,570 --> 00:18:38,220 ESI. 352 00:18:38,220 --> 00:18:40,240 So that's the one we want to look at next. 353 00:18:40,240 --> 00:18:41,580 And so that's up there. 354 00:18:41,580 --> 00:18:43,650 This is a cartoon of how this works. 355 00:18:48,570 --> 00:18:50,827 So what do you do, and how do you do this? 356 00:18:50,827 --> 00:18:53,160 So the first thing is you have your protein of interest, 357 00:18:53,160 --> 00:18:56,960 which I'll call the analyte, because we want to charge. 358 00:18:56,960 --> 00:19:00,380 Lots of times you put it under more acidic conditions, 359 00:19:00,380 --> 00:19:03,970 pH 6 or something, 6 1/2, depends on the protein. 360 00:19:03,970 --> 00:19:05,424 So you get more charge states. 361 00:19:05,424 --> 00:19:07,340 And if you're trying to look at something big, 362 00:19:07,340 --> 00:19:09,200 you need a lot of charges on there 363 00:19:09,200 --> 00:19:11,780 to get it into this mass range of 1 to 2,000 364 00:19:11,780 --> 00:19:15,060 to be able to see it using this method, 365 00:19:15,060 --> 00:19:18,290 and apparently what you do here-- 366 00:19:18,290 --> 00:19:19,810 can you see this? 367 00:19:19,810 --> 00:19:22,120 Have you done this? 368 00:19:22,120 --> 00:19:24,080 Can you see this capillary? 369 00:19:24,080 --> 00:19:25,856 Can you look at what's going on? 370 00:19:25,856 --> 00:19:26,980 AUDIENCE: I don't think so. 371 00:19:26,980 --> 00:19:27,730 JOANNE STUBBE: OK. 372 00:19:27,730 --> 00:19:30,980 So I was just wondering, because I haven't ever. 373 00:19:30,980 --> 00:19:31,987 So it's all closed off. 374 00:19:31,987 --> 00:19:34,070 It's in a box, and so you can't-- there's not like 375 00:19:34,070 --> 00:19:36,022 a thing where you can watch what's going on? 376 00:19:36,022 --> 00:19:37,230 AUDIENCE: Not that I've seen. 377 00:19:37,230 --> 00:19:40,520 JOANNE STUBBE: OK, because I think it's sort of amazing. 378 00:19:40,520 --> 00:19:43,010 How do you get this huge protein and solution 379 00:19:43,010 --> 00:19:44,710 into the gas phase? 380 00:19:44,710 --> 00:19:45,800 Right. 381 00:19:45,800 --> 00:19:49,070 I mean, that, to me, is like mind boggling, OK? 382 00:19:49,070 --> 00:19:51,270 I mean, these guys were geniuses. 383 00:19:51,270 --> 00:19:53,810 And you know, there's been a number of Nobel Prizes 384 00:19:53,810 --> 00:19:55,340 for this, but I wouldn't have a clue 385 00:19:55,340 --> 00:19:57,530 how to do something like that. 386 00:19:57,530 --> 00:20:01,970 So what you do is apparently, you put it down a capillary 387 00:20:01,970 --> 00:20:06,990 and then you spray it out, and then you have to-- 388 00:20:06,990 --> 00:20:10,340 so what you get at the end of this, this plume of spray, 389 00:20:10,340 --> 00:20:12,980 apparently you've got a lot of the analytes 390 00:20:12,980 --> 00:20:15,470 and a lot of solvent molecules, and then the goal 391 00:20:15,470 --> 00:20:18,770 is during this process, to get into the analyzer 392 00:20:18,770 --> 00:20:20,650 is to get rid of-- 393 00:20:20,650 --> 00:20:22,940 to separate all the analytes mixed together 394 00:20:22,940 --> 00:20:25,590 into a single analyte and remove all the solvent. 395 00:20:25,590 --> 00:20:27,080 OK, so that's the goal. 396 00:20:27,080 --> 00:20:28,970 And apparently, according to the people 397 00:20:28,970 --> 00:20:31,760 that were here yesterday, this is taken from, I think, 398 00:20:31,760 --> 00:20:34,067 sort of one of the papers that was first out. 399 00:20:34,067 --> 00:20:35,900 This is the way they did it in the old days. 400 00:20:35,900 --> 00:20:38,000 I don't know if they still do it this way, 401 00:20:38,000 --> 00:20:41,510 but the goal is really, to get a single analyte 402 00:20:41,510 --> 00:20:43,170 with no solvent on it. 403 00:20:43,170 --> 00:20:47,940 OK, and so the question is, how do you do this, and the chamber 404 00:20:47,940 --> 00:20:50,460 they had was at atmospheric pressure, 405 00:20:50,460 --> 00:20:54,950 and then they had a potential and pressure gradient, which 406 00:20:54,950 --> 00:21:01,640 allowed it to get into the mass, before the mass analyzer. 407 00:21:01,640 --> 00:21:03,800 So you start here with the initial spray, 408 00:21:03,800 --> 00:21:07,940 and then as you go farther, you remove some water molecules. 409 00:21:07,940 --> 00:21:10,190 You finally get to the place where 410 00:21:10,190 --> 00:21:12,080 you've removed enough water molecules 411 00:21:12,080 --> 00:21:14,820 that all these positively charged species come together, 412 00:21:14,820 --> 00:21:16,640 they're incredibly unhappy. 413 00:21:16,640 --> 00:21:18,304 And then they fragment apart. 414 00:21:18,304 --> 00:21:19,970 I mean, that's the way they describe it. 415 00:21:19,970 --> 00:21:22,046 It sounds reasonable. 416 00:21:22,046 --> 00:21:24,170 So you get smaller and smaller till eventually, you 417 00:21:24,170 --> 00:21:27,410 get to a place where you have an analyte that you can look 418 00:21:27,410 --> 00:21:31,400 at specifically and the water has been removed, 419 00:21:31,400 --> 00:21:33,791 and that's what you look at. 420 00:21:33,791 --> 00:21:34,290 OK. 421 00:21:34,290 --> 00:21:39,100 So again, we need to be in the range of 1 to 2,000. 422 00:21:39,100 --> 00:21:41,310 So that's the way these things work. 423 00:21:41,310 --> 00:21:43,710 Although, I think, again, how you 424 00:21:43,710 --> 00:21:45,960 get to looking at the single ions 425 00:21:45,960 --> 00:21:49,496 I think in different mass spectrometers. 426 00:21:49,496 --> 00:21:53,620 And so what the issues are, I think, are shown here, 427 00:21:53,620 --> 00:21:57,620 and this is the beauty of this methodology. 428 00:21:57,620 --> 00:22:00,850 So if you have a protein of 10,000 molecular weight, 429 00:22:00,850 --> 00:22:05,330 you couldn't see it, because the mass analyzer is limited. 430 00:22:05,330 --> 00:22:10,390 So you have to go all the way down to eight charges on it 431 00:22:10,390 --> 00:22:12,840 to be able to see it. 432 00:22:12,840 --> 00:22:15,180 And then, you divide that by that, 433 00:22:15,180 --> 00:22:16,480 and you get-- what do you have? 434 00:22:16,480 --> 00:22:18,146 You have to do some corrections, but you 435 00:22:18,146 --> 00:22:21,600 get something that's this size. 436 00:22:21,600 --> 00:22:24,300 OK, but you can see it now because of all the charges 437 00:22:24,300 --> 00:22:27,540 on it, but the beauty is if you add more charges, 438 00:22:27,540 --> 00:22:30,130 you get another peak. 439 00:22:30,130 --> 00:22:31,200 And you get another peak. 440 00:22:31,200 --> 00:22:32,800 And it all has the same information, 441 00:22:32,800 --> 00:22:36,046 and it just differs by the number of charge. 442 00:22:36,046 --> 00:22:37,420 So you have all this information. 443 00:22:37,420 --> 00:22:40,170 You can use that-- all these informations together 444 00:22:40,170 --> 00:22:44,230 to give you a very accurate mass on this system. 445 00:22:44,230 --> 00:22:49,480 So this method by analyzing all the data, and now the computers 446 00:22:49,480 --> 00:22:55,490 do this, I guess, routinely can give you a very accurate mass. 447 00:22:55,490 --> 00:23:01,570 So if you look at this printout, it doesn't look like that. 448 00:23:01,570 --> 00:23:03,640 This is what it looks like. 449 00:23:03,640 --> 00:23:07,020 And what do you think's going on there? 450 00:23:07,020 --> 00:23:10,480 So we look at mass charge, and we're 451 00:23:10,480 --> 00:23:17,000 in the range of 1,000 to 2,000 daltons. 452 00:23:17,000 --> 00:23:19,590 And then what is this all-- 453 00:23:19,590 --> 00:23:21,790 what is all of these peaks associated with? 454 00:23:21,790 --> 00:23:23,632 Anybody got a clue? 455 00:23:23,632 --> 00:23:24,836 AUDIENCE: Isotopes. 456 00:23:24,836 --> 00:23:26,210 JOANNE STUBBE: Yeah, so isotopes. 457 00:23:26,210 --> 00:23:29,920 So where are we seeing isotopes before? 458 00:23:29,920 --> 00:23:33,150 So these are mostly stable isotopes. 459 00:23:33,150 --> 00:23:35,630 We spent recitation two and three 460 00:23:35,630 --> 00:23:37,780 looking at radio isotopes. 461 00:23:37,780 --> 00:23:38,440 OK. 462 00:23:38,440 --> 00:23:40,360 I would say, you know, radioactivity 463 00:23:40,360 --> 00:23:41,650 is pretty important. 464 00:23:41,650 --> 00:23:45,080 Stable isotopes are extremely important to mass spectrometry. 465 00:23:45,080 --> 00:23:47,950 So if you get into this, you're going to be able-- 466 00:23:47,950 --> 00:23:50,560 you'll see that being able to label things 467 00:23:50,560 --> 00:23:52,360 with different kinds of stable isotopes 468 00:23:52,360 --> 00:23:55,660 is key to really deconvoluting the complexity when 469 00:23:55,660 --> 00:23:58,180 you're looking at a whole proteome and thousands 470 00:23:58,180 --> 00:23:59,590 of peptides. 471 00:23:59,590 --> 00:24:01,900 We're getting down-- it becomes very complicated, 472 00:24:01,900 --> 00:24:04,720 and you have to be able to compute 473 00:24:04,720 --> 00:24:09,520 what you expect based on the normal natural abundance 474 00:24:09,520 --> 00:24:11,350 isotopic distribution. 475 00:24:11,350 --> 00:24:12,770 So that's the key thing. 476 00:24:12,770 --> 00:24:18,220 So we look at the normal isotopic distribution. 477 00:24:23,970 --> 00:24:27,200 And if you look at that, I think in the next one, 478 00:24:27,200 --> 00:24:29,430 I show you an example of that. 479 00:24:29,430 --> 00:24:32,990 So what are the isotopes-- you probably can't read this here, 480 00:24:32,990 --> 00:24:36,260 but if you pull out your computer, you'll see this. 481 00:24:36,260 --> 00:24:38,410 So we have C12, C13. 482 00:24:38,410 --> 00:24:41,930 OK, we have hundreds of amino acids with carbons. 483 00:24:41,930 --> 00:24:47,006 So you have C12 and C13. 484 00:24:47,006 --> 00:24:50,170 C12 is 99%. 485 00:24:50,170 --> 00:24:51,710 C13 is 1%. 486 00:24:51,710 --> 00:24:53,090 That's an actual abundance. 487 00:24:53,090 --> 00:24:56,570 OK, so every one of these has different natural abundance. 488 00:24:56,570 --> 00:24:58,227 We know what they are. 489 00:24:58,227 --> 00:24:59,810 In fact, if you're an organic chemist, 490 00:24:59,810 --> 00:25:03,140 you can measure isotope of x using a mass spectrometer, 491 00:25:03,140 --> 00:25:05,990 if you have something that's really accurate, which we do. 492 00:25:05,990 --> 00:25:08,930 I've measured a lot of C13 isotope effects, 493 00:25:08,930 --> 00:25:13,250 using a mass spectrometer, based on differences 494 00:25:13,250 --> 00:25:15,020 in natural abundance and changes. 495 00:25:15,020 --> 00:25:16,084 Yeah. 496 00:25:16,084 --> 00:25:16,958 AUDIENCE: [INAUDIBLE] 497 00:25:16,958 --> 00:25:18,243 JOANNE STUBBE: The what? 498 00:25:18,243 --> 00:25:19,230 AUDIENCE: The natural abundance of deuterium? 499 00:25:19,230 --> 00:25:20,980 JOANNE STUBBE: Yeah, I think it's up here. 500 00:25:20,980 --> 00:25:21,860 So it's up here. 501 00:25:21,860 --> 00:25:26,842 I think it's-- let's see, 3%. 502 00:25:26,842 --> 00:25:29,113 Yeah, protons deuterium 3%. 503 00:25:29,113 --> 00:25:34,152 AUDIENCE: Would you expect a huge distribution from that? 504 00:25:34,152 --> 00:25:36,360 JOANNE STUBBE: You see isotope effects on everything. 505 00:25:36,360 --> 00:25:38,790 You see-- if you do mass spec, I mean, 506 00:25:38,790 --> 00:25:41,430 this is something I think that's not appreciated, 507 00:25:41,430 --> 00:25:44,820 and you have a linker with deuteriums in it, 508 00:25:44,820 --> 00:25:46,850 and even if you chromatograph it, 509 00:25:46,850 --> 00:25:49,440 you change the chromatographic properties based 510 00:25:49,440 --> 00:25:53,340 on the deuterium, and so you might think it's migrating 511 00:25:53,340 --> 00:25:54,390 here, and it doesn't. 512 00:25:54,390 --> 00:25:56,910 It has an isotope effect on how it migrates. 513 00:25:56,910 --> 00:26:00,000 So yeah, you need to pay attention to all of this stuff. 514 00:26:00,000 --> 00:26:02,010 OK, and it seems like a small amount, 515 00:26:02,010 --> 00:26:04,980 but the beauty is that it is a small amount, 516 00:26:04,980 --> 00:26:06,660 but it's incredibly informative, and we 517 00:26:06,660 --> 00:26:08,820 have very powerful computers that can 518 00:26:08,820 --> 00:26:10,990 allow us to do the analysis. 519 00:26:10,990 --> 00:26:13,290 So we do have protons. 520 00:26:13,290 --> 00:26:15,090 You see deuterium used. 521 00:26:15,090 --> 00:26:18,090 You saw deuterium used in this paper you read today. 522 00:26:18,090 --> 00:26:20,730 They did CD3 and CH3's. 523 00:26:20,730 --> 00:26:24,080 OK, you can also see the tritium. 524 00:26:24,080 --> 00:26:25,819 OK, that's much smaller. 525 00:26:25,819 --> 00:26:28,110 I don't know what the ratio is, but you can look at it. 526 00:26:28,110 --> 00:26:32,190 But you also-- this one is also incredibly important and is 527 00:26:32,190 --> 00:26:34,020 widely used in proteomics-- 528 00:26:34,020 --> 00:26:39,120 N14 and 15, and people do isotopic labeling. 529 00:26:39,120 --> 00:26:44,590 So they might see N15 labeled lysine or arginine 530 00:26:44,590 --> 00:26:46,890 or deuterated lysine or arginine. 531 00:26:46,890 --> 00:26:49,050 And why do you think they would deuterate 532 00:26:49,050 --> 00:26:53,070 the lysine or the arginine or N15 label it? 533 00:26:53,070 --> 00:26:55,170 What do we know about lysine and arginine 534 00:26:55,170 --> 00:26:57,690 in terms of thinking about proteins 535 00:26:57,690 --> 00:26:59,040 and analysis of proteins? 536 00:27:01,600 --> 00:27:04,790 What do you think about lysine and arginine? 537 00:27:04,790 --> 00:27:07,570 You've seen it several times over the course 538 00:27:07,570 --> 00:27:10,220 of this semester, and you probably saw it in 5.07. 539 00:27:10,220 --> 00:27:11,827 AUDIENCE: [INAUDIBLE] 540 00:27:11,827 --> 00:27:12,660 JOANNE STUBBE: What? 541 00:27:12,660 --> 00:27:14,360 AUDIENCE: The protons will exchange? 542 00:27:14,360 --> 00:27:16,193 JOANNE STUBBE: Well, now as you put it-- no. 543 00:27:16,193 --> 00:27:19,840 So that that's true if it was on a hydrogen and a nitrogen, 544 00:27:19,840 --> 00:27:23,230 it would exchange, but they put the deuteriums in on carbon, 545 00:27:23,230 --> 00:27:25,080 so they're not exchanging. 546 00:27:25,080 --> 00:27:28,600 OK, so why that would happen in any amino acid, 547 00:27:28,600 --> 00:27:30,220 why lysine and arginine? 548 00:27:30,220 --> 00:27:33,970 And the reason is that almost all-- and this was also 549 00:27:33,970 --> 00:27:38,040 done in this paper, you don't work on the huge protein. 550 00:27:38,040 --> 00:27:40,100 You cleave it to pieces. 551 00:27:40,100 --> 00:27:43,910 And you cleave it into pieces, and where you cleave 552 00:27:43,910 --> 00:27:46,510 is with trypsin, which is the major-- 553 00:27:46,510 --> 00:27:48,694 you've seen this used now over and over again. 554 00:27:48,694 --> 00:27:50,860 That's a major thing you use because it cleaves next 555 00:27:50,860 --> 00:27:54,440 to basic amino acids. 556 00:27:54,440 --> 00:27:57,550 So these become really important in labeling experiments, 557 00:27:57,550 --> 00:27:59,830 if you read much mass spec data, or if you 558 00:27:59,830 --> 00:28:02,470 look at Alice Ting's work, everything 559 00:28:02,470 --> 00:28:06,130 is N15 and deuterium labeled, and lysine 560 00:28:06,130 --> 00:28:09,130 and arginine to try to make sure they have coverage 561 00:28:09,130 --> 00:28:13,930 of the whole proteome, which is what her lab actually looks at. 562 00:28:13,930 --> 00:28:14,470 OK. 563 00:28:14,470 --> 00:28:17,800 So we have isotopic labels, and we can take advantage of these, 564 00:28:17,800 --> 00:28:21,550 and we can calculate what the distribution should 565 00:28:21,550 --> 00:28:23,650 look like, OK, of the isotopes should be, 566 00:28:23,650 --> 00:28:25,270 depending on what the-- 567 00:28:25,270 --> 00:28:26,950 we know what the sequence is. 568 00:28:26,950 --> 00:28:28,270 We know what the abundance is. 569 00:28:28,270 --> 00:28:31,135 And so you can calculate the whole mass spec. 570 00:28:34,630 --> 00:28:38,616 So let's see. 571 00:28:38,616 --> 00:28:42,970 So there's going to be a number of things that we want to do, 572 00:28:42,970 --> 00:28:47,110 and what we're going to be describing today 573 00:28:47,110 --> 00:28:52,900 and the next time is a "workflow." 574 00:28:52,900 --> 00:28:56,322 These are the words that people use all the time, 575 00:28:56,322 --> 00:28:57,090 and "platform." 576 00:29:01,630 --> 00:29:06,550 And what we're trying to do in the case of the Carroll papers 577 00:29:06,550 --> 00:29:13,111 is simply look at whether the protein is modified or not. 578 00:29:13,111 --> 00:29:15,650 But as with most post-translational 579 00:29:15,650 --> 00:29:20,350 modifications, do you think this is going to be 100% modified? 580 00:29:20,350 --> 00:29:20,850 No. 581 00:29:20,850 --> 00:29:22,650 In fact, it's only partially modified. 582 00:29:22,650 --> 00:29:25,110 That adds to the complexity of understanding 583 00:29:25,110 --> 00:29:26,820 whether the biology is interesting 584 00:29:26,820 --> 00:29:31,470 or not, so what you have then is something that's modified 585 00:29:31,470 --> 00:29:35,110 and something that's more non-modified. 586 00:29:35,110 --> 00:29:38,515 So then the question is, how do you tell how much is modified 587 00:29:38,515 --> 00:29:40,810 and how much is non-modified? 588 00:29:40,810 --> 00:29:44,800 If this enhances the rate only a factor of two, 589 00:29:44,800 --> 00:29:49,240 and this is 99.8%, of this, are you 590 00:29:49,240 --> 00:29:53,110 ever going to be able to see an effect of this modification? 591 00:29:53,110 --> 00:29:55,630 That's the question that you have to focus on, 592 00:29:55,630 --> 00:29:59,110 and everybody and his brother is doing experiments like this. 593 00:29:59,110 --> 00:30:01,720 We will see in a second, hundreds of post translational 594 00:30:01,720 --> 00:30:05,470 modifications, and the question is what are they 595 00:30:05,470 --> 00:30:10,040 doing in terms of thinking about the biology of the system. 596 00:30:10,040 --> 00:30:12,145 OK, so what's the platform? 597 00:30:14,595 --> 00:30:16,220 What's the platform we're going to use? 598 00:30:16,220 --> 00:30:18,011 So there are two ways you can look at this. 599 00:30:18,011 --> 00:30:22,267 So we have a protein that has been modified. 600 00:30:22,267 --> 00:30:24,100 You're going to-- if you had a huge protein, 601 00:30:24,100 --> 00:30:28,180 and you only had a single OH on it, even if it was 100%, 602 00:30:28,180 --> 00:30:31,784 and the protein was, say, 300,000 molecular weight, you 603 00:30:31,784 --> 00:30:32,950 might not be able to see it. 604 00:30:32,950 --> 00:30:35,520 You need to do a calculation to see whether you could see it 605 00:30:35,520 --> 00:30:36,020 or not. 606 00:30:36,020 --> 00:30:38,530 If you have a small protein of molecular weight 607 00:30:38,530 --> 00:30:40,220 30,000, or whatever-- 608 00:30:40,220 --> 00:30:43,990 I think the 22,000 or 23,000 like glutathione peroxidase, 609 00:30:43,990 --> 00:30:45,970 used in this paper, you could see it. 610 00:30:45,970 --> 00:30:47,920 So you could look at the protein directly. 611 00:30:47,920 --> 00:30:50,080 But how else could you do this? 612 00:30:50,080 --> 00:30:52,392 You would enrich. 613 00:30:52,392 --> 00:30:54,100 If you were doing this in the whole cell, 614 00:30:54,100 --> 00:30:58,770 you would want to separate this away from everything else. 615 00:30:58,770 --> 00:31:01,930 OK, so to do that, you want to be 616 00:31:01,930 --> 00:31:05,980 able to have a way to stabilize this, OK, and that's 617 00:31:05,980 --> 00:31:08,410 what this paper is all about, and then not 618 00:31:08,410 --> 00:31:11,340 only to stabilize it, but to separate 619 00:31:11,340 --> 00:31:13,000 the stabilized form out. 620 00:31:13,000 --> 00:31:15,910 So where does this happen? 621 00:31:15,910 --> 00:31:19,720 And in this particular cartoon, where do you 622 00:31:19,720 --> 00:31:22,360 see post translational modifications? 623 00:31:22,360 --> 00:31:25,330 Probably the most popular one is phosphorylation. 624 00:31:28,480 --> 00:31:31,520 So we have signaling cascades in kinases. 625 00:31:31,520 --> 00:31:34,270 And in fact, if you look at the epidermal growth factor 626 00:31:34,270 --> 00:31:36,670 receptor, it's a tyrosine kinase, 627 00:31:36,670 --> 00:31:39,440 and it gets phosphorylated and is regulated. 628 00:31:39,440 --> 00:31:42,160 And this sulfenylation is supposed 629 00:31:42,160 --> 00:31:44,835 to be on top of the phosphorylation. 630 00:31:44,835 --> 00:31:47,260 So you have multiple post-translational 631 00:31:47,260 --> 00:31:50,140 modifications that can affect activity. 632 00:31:50,140 --> 00:31:54,280 So Forest White, for example, in BE, 633 00:31:54,280 --> 00:31:57,660 works on kinase signaling cascades. 634 00:31:57,660 --> 00:32:01,180 And so he's developed a method, as have others, 635 00:32:01,180 --> 00:32:06,040 to be able to pull phosphorylated proteins out 636 00:32:06,040 --> 00:32:08,061 of a crude gemisch. 637 00:32:08,061 --> 00:32:08,560 OK. 638 00:32:08,560 --> 00:32:10,860 So, you know, if you look at this, 639 00:32:10,860 --> 00:32:15,100 here he's got iron bound to a phosphate 640 00:32:15,100 --> 00:32:16,530 and bound to some bead. 641 00:32:16,530 --> 00:32:19,390 So the iron's bound to some chelate around the bead, 642 00:32:19,390 --> 00:32:21,520 just like your nickel affinity column, which 643 00:32:21,520 --> 00:32:25,270 then binds to the protein. 644 00:32:25,270 --> 00:32:29,250 But this raises the issue that I was discussing in class, which 645 00:32:29,250 --> 00:32:31,390 I spent a lot of time on over and over again, 646 00:32:31,390 --> 00:32:32,890 but you need to think about, do you 647 00:32:32,890 --> 00:32:34,652 think these bonds are tight, how tight 648 00:32:34,652 --> 00:32:35,860 do you think those bonds are? 649 00:32:38,870 --> 00:32:41,720 What do you need to think about for this kind of analysis 650 00:32:41,720 --> 00:32:44,330 to work? 651 00:32:44,330 --> 00:32:46,649 It's the same thing with nickel affinity column 652 00:32:46,649 --> 00:32:48,440 that you talked about when you were looking 653 00:32:48,440 --> 00:32:51,750 at purification of proteins. 654 00:32:51,750 --> 00:32:53,590 AUDIENCE: It has to be stable enough. 655 00:32:53,590 --> 00:32:54,850 JOANNE STUBBE: It has to be stable enough. 656 00:32:54,850 --> 00:32:55,580 That's the key. 657 00:32:55,580 --> 00:32:58,860 So you have to undergo ligand exchange. 658 00:32:58,860 --> 00:33:00,680 It's got to-- if you didn't have-- 659 00:33:00,680 --> 00:33:03,000 when you start, you don't have phosphorylated 660 00:33:03,000 --> 00:33:04,340 form of your protein around. 661 00:33:04,340 --> 00:33:05,048 You have nothing. 662 00:33:05,048 --> 00:33:06,280 You have water there. 663 00:33:06,280 --> 00:33:09,630 OK, so the waters have to undergo exchange, 664 00:33:09,630 --> 00:33:13,520 so the phosphate can then bind, but it's an equilibrium, 665 00:33:13,520 --> 00:33:16,680 and so up and down the column is coming off and on. 666 00:33:16,680 --> 00:33:17,494 Yeah. 667 00:33:17,494 --> 00:33:23,461 AUDIENCE: [INAUDIBLE] 668 00:33:23,461 --> 00:33:24,460 JOANNE STUBBE: It could. 669 00:33:24,460 --> 00:33:26,668 I mean, so it's a question of what out competes what. 670 00:33:26,668 --> 00:33:28,470 It's a question of relative Kds. 671 00:33:28,470 --> 00:33:31,110 So what you have to do is study all of this 672 00:33:31,110 --> 00:33:33,424 to figure out how to optimize this, 673 00:33:33,424 --> 00:33:34,590 how did they arrive at this? 674 00:33:34,590 --> 00:33:36,380 Probably somebody did a lot of studies. 675 00:33:36,380 --> 00:33:37,710 OK. 676 00:33:37,710 --> 00:33:38,850 This is a new method. 677 00:33:38,850 --> 00:33:40,350 I don't know how new it is, but it's 678 00:33:40,350 --> 00:33:43,890 a method I don't know that much about, again, of pulling 679 00:33:43,890 --> 00:33:45,240 phosphates out. 680 00:33:45,240 --> 00:33:47,220 So that's one way. 681 00:33:47,220 --> 00:33:51,780 So you have-- so you usually have an affinity purification. 682 00:33:56,350 --> 00:33:59,680 And if we look at the Carroll paper, what she does 683 00:33:59,680 --> 00:34:02,200 in the next paper is she's going to figure out a way-- 684 00:34:02,200 --> 00:34:06,130 she's derivatized, she's made a dimedone derivative, which 685 00:34:06,130 --> 00:34:08,980 stabilizes the sulfenic acid, and then 686 00:34:08,980 --> 00:34:10,630 she attaches something to it that's 687 00:34:10,630 --> 00:34:12,850 going to allow us to affinity purify that. 688 00:34:12,850 --> 00:34:15,370 We'll come back and talk about that later. 689 00:34:15,370 --> 00:34:19,000 So what are they using over here? 690 00:34:19,000 --> 00:34:22,389 They're using-- this is-- if you look at histones that 691 00:34:22,389 --> 00:34:25,239 get acetylated or methylated, they 692 00:34:25,239 --> 00:34:28,540 have an antibody that's specific for the acetylated lysine, 693 00:34:28,540 --> 00:34:31,060 so they use antibodies to pull something out. 694 00:34:31,060 --> 00:34:34,389 So that's a method-- the second way of pulling things out 695 00:34:34,389 --> 00:34:35,870 are using antibodies. 696 00:34:35,870 --> 00:34:37,480 That's quite frequently used. 697 00:34:37,480 --> 00:34:40,190 And what did they use in this paper? 698 00:34:40,190 --> 00:34:44,184 Did it detect the modified sulfenic acid? 699 00:34:44,184 --> 00:34:45,100 Does anybody remember? 700 00:34:45,100 --> 00:34:47,132 Did you read the paper carefully enough? 701 00:34:47,132 --> 00:34:48,840 AUDIENCE: Like, a anti-dimedone antibody? 702 00:34:48,840 --> 00:34:51,770 JOANNE STUBBE: Yeah, so they use an antidimedone antibody. 703 00:34:51,770 --> 00:34:53,770 OK, so that becomes really critical 704 00:34:53,770 --> 00:34:56,860 that you know that your antibodies are actually 705 00:34:56,860 --> 00:34:58,870 working effectively. 706 00:34:58,870 --> 00:35:01,600 So we have antibodies, and then, another thing 707 00:35:01,600 --> 00:35:05,080 that people are interested in this department, the Imperiali 708 00:35:05,080 --> 00:35:08,560 lab, is sugars. 709 00:35:08,560 --> 00:35:10,230 We have sugars everywhere. 710 00:35:10,230 --> 00:35:13,360 OK, we don't really understand the function of these sugars. 711 00:35:13,360 --> 00:35:17,390 We understand some of them, but it's amazingly complex. 712 00:35:17,390 --> 00:35:21,040 And what we have are proteins called lectins, and any of you 713 00:35:21,040 --> 00:35:22,896 heard Laura Kiessling talk, maybe 714 00:35:22,896 --> 00:35:24,520 undergraduates wouldn't have done this, 715 00:35:24,520 --> 00:35:28,750 but she discovered a new lectin and discovered 716 00:35:28,750 --> 00:35:30,610 the basis, the structure the sugar 717 00:35:30,610 --> 00:35:31,960 that binds to this lectin. 718 00:35:31,960 --> 00:35:35,590 And so you can selectively move that type of sugar. 719 00:35:35,590 --> 00:35:36,800 Again, it's an equilibrium. 720 00:35:36,800 --> 00:35:39,760 So they're coming off and on, but it binds, hopefully, 721 00:35:39,760 --> 00:35:42,310 enough so that the other stuff washes through, 722 00:35:42,310 --> 00:35:46,030 and you enrich in the protein of interest. 723 00:35:46,030 --> 00:35:49,510 So these are sort of some of the tricks that are actually used. 724 00:35:49,510 --> 00:35:52,360 We're going to see, in the case of the Carroll paper, 725 00:35:52,360 --> 00:35:55,840 next time we use click chemistry to make something 726 00:35:55,840 --> 00:35:58,210 with a biotin on it, because biotin you all know 727 00:35:58,210 --> 00:36:02,590 can bind to streptavidin, which has pluses, and it has minuses, 728 00:36:02,590 --> 00:36:04,090 but it allows you to pull things out 729 00:36:04,090 --> 00:36:08,170 more easily, because the interaction is so tight. 730 00:36:08,170 --> 00:36:10,885 So you could do this-- 731 00:36:10,885 --> 00:36:15,930 the workflow could be on the intact protein, 732 00:36:15,930 --> 00:36:21,310 or it could be on peptides. 733 00:36:21,310 --> 00:36:22,630 OK. 734 00:36:22,630 --> 00:36:26,510 And so the bottom half of this graph 735 00:36:26,510 --> 00:36:29,772 shows what happens after you treat this with trypsin. 736 00:36:29,772 --> 00:36:32,090 So with trypsin, and you're always cleaving next 737 00:36:32,090 --> 00:36:33,360 to lysine or arginine. 738 00:36:33,360 --> 00:36:35,060 So the C terminus of your protein 739 00:36:35,060 --> 00:36:38,310 is always a lysine or an arginine. 740 00:36:38,310 --> 00:36:41,990 And you can find that more easily if you deuterate 741 00:36:41,990 --> 00:36:43,010 or N15 label it. 742 00:36:43,010 --> 00:36:46,090 That's what people routinely do in the [? Broad. ?] 743 00:36:46,090 --> 00:36:48,920 And then you have, I think this is the most amazing thing, 744 00:36:48,920 --> 00:36:50,430 so you have a protein. 745 00:36:50,430 --> 00:36:52,250 And then you have an HPLC column. 746 00:36:52,250 --> 00:36:54,710 Have any of you done HPLC? 747 00:36:54,710 --> 00:36:57,560 And so do you think-- 748 00:36:57,560 --> 00:37:00,380 you could have a protein of 300,000 molecular weight, 749 00:37:00,380 --> 00:37:04,120 and look at the separation of your peptides. 750 00:37:04,120 --> 00:37:06,000 But if you look at any one of these things, 751 00:37:06,000 --> 00:37:08,890 do you think it's pure? 752 00:37:08,890 --> 00:37:09,760 So it's not pure. 753 00:37:09,760 --> 00:37:13,030 So every one of these peaks, if it's 300,000 molecular weight, 754 00:37:13,030 --> 00:37:14,200 you can calculate-- 755 00:37:14,200 --> 00:37:16,270 the reason people use trypsin is-- 756 00:37:16,270 --> 00:37:18,270 does anybody know why use trypsin, besides that 757 00:37:18,270 --> 00:37:20,820 cleaves at lysines in its specifics? 758 00:37:20,820 --> 00:37:23,200 Why do people use trypsin as a thing 759 00:37:23,200 --> 00:37:25,360 to cleave a big thing down into a little thing? 760 00:37:28,122 --> 00:37:30,080 AUDIENCE: What's the rationale for cleaving it? 761 00:37:30,080 --> 00:37:30,897 [INAUDIBLE] 762 00:37:30,897 --> 00:37:32,730 JOANNE STUBBE: So the rationale for cleaving 763 00:37:32,730 --> 00:37:35,224 it is just to make it smaller and easier to analyze. 764 00:37:35,224 --> 00:37:36,765 That's the rationale for cleaving it. 765 00:37:36,765 --> 00:37:38,850 So a peptide, a small peptide. 766 00:37:38,850 --> 00:37:42,420 But the question is, how big is the small peptide 767 00:37:42,420 --> 00:37:43,960 that's easy to analyze? 768 00:37:43,960 --> 00:37:45,360 And so that's the rationale. 769 00:37:45,360 --> 00:37:48,990 It gives you a distribution of peptides that's pretty good, 770 00:37:48,990 --> 00:37:52,140 that are all accessible to mass spec methods. 771 00:37:52,140 --> 00:37:54,660 So I don't know what the distribution is, but you know, 772 00:37:54,660 --> 00:37:56,670 people have done that calculation. 773 00:37:56,670 --> 00:37:58,490 And so almost always the peptides fly, 774 00:37:58,490 --> 00:38:00,990 whereas if you use other things, and you have something much 775 00:38:00,990 --> 00:38:04,980 bigger, it might not get ionized in the appropriate way 776 00:38:04,980 --> 00:38:09,090 or in a quantitative way, and you completely miss it. 777 00:38:09,090 --> 00:38:11,280 So the trypsin has been most successful. 778 00:38:11,280 --> 00:38:15,900 But each one of these little peaks is not one peak. 779 00:38:15,900 --> 00:38:19,350 You'll see when you put it into the mass analyzer, 780 00:38:19,350 --> 00:38:21,030 and if you read this paper carefully, 781 00:38:21,030 --> 00:38:23,790 you will see they got multiple mass charge 782 00:38:23,790 --> 00:38:26,040 species, which then they associated 783 00:38:26,040 --> 00:38:28,470 with specific peptides, OK. 784 00:38:28,470 --> 00:38:30,780 They know the sequence of their protein. 785 00:38:30,780 --> 00:38:34,920 And then they always use tosyl phenyl chloro ketone. 786 00:38:34,920 --> 00:38:35,940 Why do they use that? 787 00:38:35,940 --> 00:38:37,482 Anybody have an any idea? 788 00:38:37,482 --> 00:38:38,940 So in the experiments where they're 789 00:38:38,940 --> 00:38:43,570 doing the trypsin cleavage, they put in tosyl phenyl chloro 790 00:38:43,570 --> 00:38:44,250 ketone. 791 00:38:44,250 --> 00:38:45,150 Anybody know why? 792 00:38:51,700 --> 00:38:52,200 OK. 793 00:38:52,200 --> 00:38:52,870 No good. 794 00:38:52,870 --> 00:38:54,590 This is something that-- 795 00:38:54,590 --> 00:38:58,240 so tosyl phenyl chloro ketone is an alpha halo ketone. 796 00:38:58,240 --> 00:39:00,560 So it's activated for nucleophilic attack, 797 00:39:00,560 --> 00:39:03,820 and what you do is you have an acylated N 798 00:39:03,820 --> 00:39:06,970 terminus and an aromatic, and that's specific 799 00:39:06,970 --> 00:39:09,910 for chymotrypsin, like proteases. 800 00:39:09,910 --> 00:39:13,000 And so what this does is that covalently 801 00:39:13,000 --> 00:39:16,960 modifies the active site of chymotrypsin, 802 00:39:16,960 --> 00:39:20,340 and kills chymotrypsin. 803 00:39:20,340 --> 00:39:23,950 If you choose the wrong time to cleave with trypsin, 804 00:39:23,950 --> 00:39:26,460 you don't start getting cleavage next to hydrophobics, 805 00:39:26,460 --> 00:39:30,722 which then makes the analysis of the peptides much more complex. 806 00:39:30,722 --> 00:39:32,680 So the analysis of the peptide, a lot of people 807 00:39:32,680 --> 00:39:34,960 have done a lot of peptide chemistry, 808 00:39:34,960 --> 00:39:36,800 and I was telling this story before. 809 00:39:36,800 --> 00:39:38,170 I always go off on tangents. 810 00:39:38,170 --> 00:39:40,269 But Stein and Moore won the Nobel Prize. 811 00:39:40,269 --> 00:39:42,060 Maybe this is what you do when you get old, 812 00:39:42,060 --> 00:39:46,810 but Stein and Moore won the Nobel Prize, 813 00:39:46,810 --> 00:39:49,360 you know, in the 1950s, the 1950s, 814 00:39:49,360 --> 00:39:51,370 for separating amino acids. 815 00:39:51,370 --> 00:39:55,570 Do you know that they had a three story column of Dowex 816 00:39:55,570 --> 00:39:59,200 that was composed of anion exchange Dowex and cations? 817 00:39:59,200 --> 00:40:02,770 It was all polystyrene backbones of anion and cation 818 00:40:02,770 --> 00:40:06,200 polystyrenes, to be able to separate the amino acids. 819 00:40:06,200 --> 00:40:06,820 OK. 820 00:40:06,820 --> 00:40:09,550 And when you do that, of course, it gets stuck on the resin. 821 00:40:09,550 --> 00:40:12,820 Your recovery's out of the bottom of this chromatography. 822 00:40:12,820 --> 00:40:16,424 You need tons of stuff to put on the column in the first place. 823 00:40:16,424 --> 00:40:17,590 And this is what's happened. 824 00:40:17,590 --> 00:40:20,500 I mean, you have a little tiny HPLC column 825 00:40:20,500 --> 00:40:23,020 that has huge number of theoretical place 826 00:40:23,020 --> 00:40:25,540 that allows you amazing separations. 827 00:40:25,540 --> 00:40:29,980 I mean, again, the technology is sort of mind boggling, what 828 00:40:29,980 --> 00:40:30,830 you can do now. 829 00:40:30,830 --> 00:40:34,356 OK, so what you're doing here is then 830 00:40:34,356 --> 00:40:35,980 you're just asking the question, if you 831 00:40:35,980 --> 00:40:39,430 have a post-translational modification, x, 832 00:40:39,430 --> 00:40:41,665 you can either look at the entire protein. 833 00:40:45,440 --> 00:40:47,550 And so you could probably tell it was modified, 834 00:40:47,550 --> 00:40:50,037 but telling the location of the modified location, 835 00:40:50,037 --> 00:40:51,870 you can't, or you can treat it with trypsin. 836 00:40:54,374 --> 00:40:56,970 And then you get, again, with trypsin, 837 00:40:56,970 --> 00:40:58,790 you have little pieces. 838 00:40:58,790 --> 00:41:02,770 And one of these little pieces will have an x on it. 839 00:41:02,770 --> 00:41:04,010 And then you can define it. 840 00:41:04,010 --> 00:41:06,840 And then if you want to do sophisticated analysis, 841 00:41:06,840 --> 00:41:07,800 you can hit it-- 842 00:41:07,800 --> 00:41:11,760 use a second mass spectrometer, and actually sequence this. 843 00:41:11,760 --> 00:41:12,540 OK. 844 00:41:12,540 --> 00:41:17,540 So I think the next one just briefly goes to MALDI. 845 00:41:17,540 --> 00:41:20,850 And MALDI-- so Matrix Assistant Laser Disorption-- 846 00:41:20,850 --> 00:41:23,350 have any of you ever done that? 847 00:41:23,350 --> 00:41:23,850 OK. 848 00:41:23,850 --> 00:41:25,420 So where do you do that? 849 00:41:25,420 --> 00:41:26,892 Do you do that in [INAUDIBLE] lab? 850 00:41:26,892 --> 00:41:28,350 AUDIENCE: No, in the undergrad lab. 851 00:41:28,350 --> 00:41:30,683 JOANNE STUBBE: Oh, OK, because this is Brad's new thing. 852 00:41:30,683 --> 00:41:31,700 OK. 853 00:41:31,700 --> 00:41:34,600 OK, so you're looking at peptides. 854 00:41:34,600 --> 00:41:40,284 OK, so what do you use as the matrix? 855 00:41:40,284 --> 00:41:42,022 AUDIENCE: We used some aromatic acid. 856 00:41:42,022 --> 00:41:42,730 I don't remember. 857 00:41:42,730 --> 00:41:43,479 JOANNE STUBBE: OK. 858 00:41:43,479 --> 00:41:46,730 So you probably used sinapinic acid. 859 00:41:46,730 --> 00:41:49,060 AUDIENCE: [INAUDIBLE] 860 00:41:49,060 --> 00:41:49,810 JOANNE STUBBE: OK. 861 00:41:49,810 --> 00:41:53,250 So this is so-- you're using a different one still from this 862 00:41:53,250 --> 00:41:53,890 one-- this is-- 863 00:41:53,890 --> 00:41:54,270 I don't know. 864 00:41:54,270 --> 00:41:55,353 I got this idea somewhere. 865 00:41:55,353 --> 00:41:56,100 I don't know. 866 00:41:56,100 --> 00:41:57,120 So when I've done this-- 867 00:41:57,120 --> 00:41:58,830 I did do this maybe 10 years ago-- 868 00:41:58,830 --> 00:42:02,250 I've looked at a lot of peptides. 869 00:42:02,250 --> 00:42:03,870 We went through five or six of them 870 00:42:03,870 --> 00:42:05,770 before we found one that really worked well. 871 00:42:05,770 --> 00:42:07,800 So I don't know how state of the art has become, 872 00:42:07,800 --> 00:42:09,490 you know what it is. 873 00:42:09,490 --> 00:42:12,150 But the other one in the book that I got this from 874 00:42:12,150 --> 00:42:14,860 was, again, an acid. 875 00:42:14,860 --> 00:42:16,720 And so what is the idea? 876 00:42:16,720 --> 00:42:20,760 So the first thing you have to do is you have to ionize. 877 00:42:20,760 --> 00:42:27,150 So the way you do that is you mix your matrix and solution 878 00:42:27,150 --> 00:42:29,850 with your protein of interest, your analyte, 879 00:42:29,850 --> 00:42:31,230 then you evaporate it. 880 00:42:31,230 --> 00:42:33,840 So you have a solid on a little plate. 881 00:42:33,840 --> 00:42:38,580 And then you use a laser beam at 337 nanometers. 882 00:42:38,580 --> 00:42:41,520 And the light is absorbed by whatever 883 00:42:41,520 --> 00:42:46,050 the matrix is and causes you to have a plume of material. 884 00:42:46,050 --> 00:42:49,110 This is, again, amazing to me that the protein 885 00:42:49,110 --> 00:42:51,940 goes into the gas phase. 886 00:42:51,940 --> 00:42:57,960 And then, you have to go through this, go into the analyzer. 887 00:42:57,960 --> 00:42:59,369 Did you do time of flight? 888 00:42:59,369 --> 00:43:00,660 OK, so you have time of flight. 889 00:43:00,660 --> 00:43:02,520 So you guys know what it is then. 890 00:43:02,520 --> 00:43:04,530 And in the end, you do detection. 891 00:43:04,530 --> 00:43:07,210 So, again, the protocol is the same, 892 00:43:07,210 --> 00:43:13,260 but the method is different, and this is widely used and easy 893 00:43:13,260 --> 00:43:16,420 really easy to use nowadays. 894 00:43:16,420 --> 00:43:19,140 So the issue then is this is what you face when you're 895 00:43:19,140 --> 00:43:20,800 looking at a whole proteome. 896 00:43:20,800 --> 00:43:24,180 So you just can't calculate the mass of all the proteins 897 00:43:24,180 --> 00:43:25,901 from the gene sequences. 898 00:43:25,901 --> 00:43:26,400 Why? 899 00:43:26,400 --> 00:43:30,600 Because almost every single amino acid in your proteins 900 00:43:30,600 --> 00:43:32,640 are modified. 901 00:43:32,640 --> 00:43:34,920 So that adds complexity to all of this. 902 00:43:34,920 --> 00:43:39,220 So de-convoluting the mass spec becomes more complicated. 903 00:43:39,220 --> 00:43:42,130 So this just shows you, you don't need to look at this, 904 00:43:42,130 --> 00:43:45,930 but if you look at cystine, you could form disulfides. 905 00:43:45,930 --> 00:43:49,710 You can attach a prenyl group, an isoprene group on it. 906 00:43:49,710 --> 00:43:52,320 You can attatch palmitic acid on it. 907 00:43:52,320 --> 00:43:54,210 You can sulfenylate it. 908 00:43:54,210 --> 00:43:56,390 You can nitrosate it. 909 00:43:56,390 --> 00:43:59,100 So you have many, many modifications 910 00:43:59,100 --> 00:44:02,040 of the amino acids that are chemically 911 00:44:02,040 --> 00:44:04,740 reactive and involved on catalysis, 912 00:44:04,740 --> 00:44:06,440 and then not only involving catalysis, 913 00:44:06,440 --> 00:44:08,400 they are involved in regulation. 914 00:44:08,400 --> 00:44:10,470 So that then adds to the complexity 915 00:44:10,470 --> 00:44:13,800 of trying to deconvolute what the mass spec, I think, 916 00:44:13,800 --> 00:44:16,080 is actually telling you. 917 00:44:16,080 --> 00:44:18,570 And then, sorry, it went backwards. 918 00:44:18,570 --> 00:44:21,440 And so then what that does is tells you-- whoops. 919 00:44:23,980 --> 00:44:27,010 I'm just completely discombobulated here. 920 00:44:27,010 --> 00:44:29,760 OK, so what that does is that, again, you're 921 00:44:29,760 --> 00:44:33,060 just adding different masses on to all of these amino acids. 922 00:44:33,060 --> 00:44:36,650 The problem is that you have modified, 923 00:44:36,650 --> 00:44:39,120 and you have unmodified. 924 00:44:39,120 --> 00:44:41,850 And the question is what's the distribution? 925 00:44:41,850 --> 00:44:46,560 OK, and so if you have a very non-abundant protein, and most 926 00:44:46,560 --> 00:44:49,470 of it's unmodified, it's going to be much harder to find. 927 00:44:49,470 --> 00:44:51,750 So these are just things you need to think about, 928 00:44:51,750 --> 00:44:54,060 and your technology to look needs 929 00:44:54,060 --> 00:44:58,185 to be extremely well worked out, so that when you look and you 930 00:44:58,185 --> 00:45:00,060 don't find something, you know what the lower 931 00:45:00,060 --> 00:45:02,190 limits of detection are. 932 00:45:04,960 --> 00:45:07,230 So here we are at our system. 933 00:45:07,230 --> 00:45:09,970 Now we're into the Carroll paper, 934 00:45:09,970 --> 00:45:15,610 and so what we're looking at is sulfenic acids, degenerated 935 00:45:15,610 --> 00:45:17,680 by hydrogen peroxide. 936 00:45:17,680 --> 00:45:20,260 We'll see-- do you think that's a fast reaction, 937 00:45:20,260 --> 00:45:21,835 hydrogen peroxide with a cystine? 938 00:45:24,460 --> 00:45:25,660 Anybody have any intuition? 939 00:45:25,660 --> 00:45:28,330 I think these reactive oxygen species you're going to find 940 00:45:28,330 --> 00:45:31,720 are not so intuitive about the chemical reactivity. 941 00:45:31,720 --> 00:45:34,960 I'll give you a table with what we think we know in general. 942 00:45:34,960 --> 00:45:37,810 But I think it's not so intuitive. 943 00:45:37,810 --> 00:45:40,120 If you look at the rate constants for reaction 944 00:45:40,120 --> 00:45:43,450 of a hydrogen peroxide with a cystine 945 00:45:43,450 --> 00:45:47,540 it's 1 per molar per second, really slow. 946 00:45:47,540 --> 00:45:48,090 OK. 947 00:45:48,090 --> 00:45:50,220 So then the question you have to ask yourself, 948 00:45:50,220 --> 00:45:51,803 so this was something that was debated 949 00:45:51,803 --> 00:45:53,790 in the literature for 15 years. 950 00:45:53,790 --> 00:45:57,390 Is this so slow that this could never happen inside the cell? 951 00:45:57,390 --> 00:46:00,160 Because I just gave you a second order rate concept. 952 00:46:00,160 --> 00:46:01,740 So we have two molecules interacting 953 00:46:01,740 --> 00:46:04,150 at the concentration, this could be high. 954 00:46:04,150 --> 00:46:05,340 This is really low. 955 00:46:05,340 --> 00:46:06,840 You can calculate the rate constant 956 00:46:06,840 --> 00:46:08,010 for the actual reaction. 957 00:46:08,010 --> 00:46:10,720 It's really, really slow. 958 00:46:10,720 --> 00:46:14,730 OK, so we'll see that there are some proteins, 959 00:46:14,730 --> 00:46:18,630 peroxiredoxins that are in humans, 960 00:46:18,630 --> 00:46:21,300 are there in quite high levels that 961 00:46:21,300 --> 00:46:23,790 can increase this rate to 10 to the fourth per molar 962 00:46:23,790 --> 00:46:24,760 per second. 963 00:46:24,760 --> 00:46:26,940 So there's a huge rate increase but you 964 00:46:26,940 --> 00:46:29,550 need to think about all this kinetic stuff 965 00:46:29,550 --> 00:46:32,310 to really understand if this modification can 966 00:46:32,310 --> 00:46:34,380 happen inside the cell. 967 00:46:34,380 --> 00:46:35,950 Otherwise, well, if it can't happen, 968 00:46:35,950 --> 00:46:37,825 why are you wasting your time looking for it? 969 00:46:37,825 --> 00:46:41,530 Which is what a lot of people are doing scientifically. 970 00:46:41,530 --> 00:46:44,920 OK, so let me see what the next-- 971 00:46:44,920 --> 00:46:45,660 OK. 972 00:46:45,660 --> 00:46:55,900 So now we're into making a reagent that can specifically 973 00:46:55,900 --> 00:46:59,980 modify this, or specifically modify this. 974 00:46:59,980 --> 00:47:00,610 OK. 975 00:47:00,610 --> 00:47:02,650 So the reagent that they chose-- 976 00:47:02,650 --> 00:47:05,760 she didn't invent this reagent-- 977 00:47:05,760 --> 00:47:07,776 was dimedone. 978 00:47:16,530 --> 00:47:21,860 And this reagent specifically interacts with sulfenic acids. 979 00:47:21,860 --> 00:47:24,860 It doesn't react with the free cystine. 980 00:47:24,860 --> 00:47:26,870 So you've got to study all of this. 981 00:47:26,870 --> 00:47:29,950 And if you're going to use this as a reagent inside the cell, 982 00:47:29,950 --> 00:47:31,780 you want it to be fast. 983 00:47:31,780 --> 00:47:34,380 You don't want to take 30 hours to do the reaction. 984 00:47:34,380 --> 00:47:38,100 You want it to be over fast, and you want it to happen at pH 7. 985 00:47:38,100 --> 00:47:40,940 So how do you think this reaction works? 986 00:47:40,940 --> 00:47:44,649 Where's the most reactive part of this molecule? 987 00:47:44,649 --> 00:47:46,130 AUDIENCE: Those two protons? 988 00:47:46,130 --> 00:47:47,380 JOANNE STUBBE: So two protons. 989 00:47:47,380 --> 00:47:49,460 So this these have low pKas, so you can easily 990 00:47:49,460 --> 00:47:51,320 form the enolate. 991 00:47:51,320 --> 00:47:59,380 Depends on the details, the experimental details. 992 00:47:59,380 --> 00:48:08,490 And now you have this, and what you end up with 993 00:48:08,490 --> 00:48:10,300 is this molecule. 994 00:48:10,300 --> 00:48:12,780 And so the question is, does this go in 5%? 995 00:48:12,780 --> 00:48:17,010 You need something that goes in quantitative yield at pH 7, 996 00:48:17,010 --> 00:48:17,850 rapidly. 997 00:48:17,850 --> 00:48:20,130 OK, we're going to come back and talk about what the issues are, 998 00:48:20,130 --> 00:48:21,838 because the issues are even harder if you 999 00:48:21,838 --> 00:48:23,550 want this region to work inside the cell. 1000 00:48:23,550 --> 00:48:26,730 OK, we're doing this on glutathione peroxidase, which 1001 00:48:26,730 --> 00:48:31,751 is what he's using as a model to see if all of this stuff works. 1002 00:48:31,751 --> 00:48:32,250 OK. 1003 00:48:32,250 --> 00:48:34,350 So what you really want to do if you're 1004 00:48:34,350 --> 00:48:36,450 thinking about regulation in the end, 1005 00:48:36,450 --> 00:48:40,260 is you want to know how much is in each form, and you know, 1006 00:48:40,260 --> 00:48:43,350 if you read hundreds of papers published on methods trying 1007 00:48:43,350 --> 00:48:47,070 to figure this all out, but what she did in this case, 1008 00:48:47,070 --> 00:48:55,330 was she developed a second reagent with an iodo group. 1009 00:49:00,130 --> 00:49:01,560 OK. 1010 00:49:01,560 --> 00:49:04,470 And as you can see, what is the product of the reaction? 1011 00:49:04,470 --> 00:49:08,750 The product of the reaction is the same 1012 00:49:08,750 --> 00:49:11,790 as the product of this reagent. 1013 00:49:11,790 --> 00:49:15,550 But this reagent does not react with sulfenic acid. 1014 00:49:15,550 --> 00:49:17,550 OK, so you get no reaction. 1015 00:49:17,550 --> 00:49:18,890 So how does this reaction work? 1016 00:49:24,410 --> 00:49:25,215 What do you think? 1017 00:49:25,215 --> 00:49:26,490 The what? 1018 00:49:26,490 --> 00:49:27,970 AUDIENCE: SN2. 1019 00:49:27,970 --> 00:49:29,720 JOANNE STUBBE: So it could work by an SN2, 1020 00:49:29,720 --> 00:49:33,250 but the way probably works is it attacks the iodine. 1021 00:49:33,250 --> 00:49:37,370 So you form-- this is probably the mechanism from what's 1022 00:49:37,370 --> 00:49:40,210 been done in the literature. 1023 00:49:40,210 --> 00:49:45,890 So you attack this, and you form this, which then 1024 00:49:45,890 --> 00:49:47,986 gets attacked by the enolate. 1025 00:49:51,560 --> 00:49:53,750 So it doesn't really matter what the mechanism is, 1026 00:49:53,750 --> 00:49:56,450 but the key thing is for this to react-- 1027 00:49:56,450 --> 00:49:59,120 if you're interested in a mechanism, which I am, 1028 00:49:59,120 --> 00:50:02,870 it does matter what it is. 1029 00:50:02,870 --> 00:50:05,050 So the key thing is now you have the same reagents. 1030 00:50:05,050 --> 00:50:07,790 So how could you ever use it attached? 1031 00:50:07,790 --> 00:50:11,900 How could you ever use it to distinguish sulfenylation 1032 00:50:11,900 --> 00:50:13,160 from a cystine. 1033 00:50:13,160 --> 00:50:16,255 So what did they do in this paper? 1034 00:50:16,255 --> 00:50:17,130 AUDIENCE: [INAUDIBLE] 1035 00:50:17,130 --> 00:50:19,671 JOANNE STUBBE: Yeah, so they put the deuterated form on this. 1036 00:50:19,671 --> 00:50:22,580 So what they did then was in this paper, 1037 00:50:22,580 --> 00:50:26,200 so you got to keep these straight, 1038 00:50:26,200 --> 00:50:28,880 if they see deuteriums present, so they 1039 00:50:28,880 --> 00:50:31,880 made this deuterium label, and this 1040 00:50:31,880 --> 00:50:35,700 protonated so now you have a mass difference of 6. 1041 00:50:35,700 --> 00:50:36,200 OK. 1042 00:50:36,200 --> 00:50:40,170 And in the system, they're using glutathione peroxidase, 1043 00:50:40,170 --> 00:50:42,240 which has three cystines in it. 1044 00:50:42,240 --> 00:50:46,310 And one of the cystines is more reactive than the other two, 1045 00:50:46,310 --> 00:50:50,600 but for proof of concept they mutated two of these cystines 1046 00:50:50,600 --> 00:50:53,980 into serine initially, so you only had a single reactive 1047 00:50:53,980 --> 00:50:57,920 cystine, but then they went back and studied the whole protein. 1048 00:50:57,920 --> 00:51:02,760 OK, so let me just introduce you to this, 1049 00:51:02,760 --> 00:51:05,830 and then we'll come back and talk about this next time. 1050 00:51:05,830 --> 00:51:08,100 Let me just do one more thing. 1051 00:51:08,100 --> 00:51:13,290 OK, so here is the difference in mass between these two species. 1052 00:51:13,290 --> 00:51:15,420 So this is what you're looking at. 1053 00:51:15,420 --> 00:51:20,320 If they start out with deuterium labeled dimedone, 1054 00:51:20,320 --> 00:51:22,020 the peak that they observe is going 1055 00:51:22,020 --> 00:51:24,380 to be associated with sulfenylation, 1056 00:51:24,380 --> 00:51:27,410 and if they start out with the protonated material, 1057 00:51:27,410 --> 00:51:30,170 the peak they observe is going to be associated 1058 00:51:30,170 --> 00:51:32,150 with the [INAUDIBLE] group. 1059 00:51:32,150 --> 00:51:34,580 OK, so that's the idea. 1060 00:51:34,580 --> 00:51:38,970 And then what they did was they simply took their protein, 1061 00:51:38,970 --> 00:51:43,900 and they have, in this case, 50 micromolar of their protein, 1062 00:51:43,900 --> 00:51:46,711 and then they increase the concentration of hydrogen 1063 00:51:46,711 --> 00:51:47,210 peroxide. 1064 00:51:47,210 --> 00:51:48,959 They don't really talk very much about how 1065 00:51:48,959 --> 00:51:53,130 they design the timing, but they use, you know, two equivalents. 1066 00:51:53,130 --> 00:51:56,930 So they use variable amounts of hydrogen peroxide. 1067 00:51:56,930 --> 00:52:01,590 And what you can see is the maximum amount. 1068 00:52:01,590 --> 00:52:03,920 So now what you're using, we talked about this before, 1069 00:52:03,920 --> 00:52:09,380 but we're using anti dimedone antibodies for the detection. 1070 00:52:09,380 --> 00:52:14,580 And here, they're starting with no hydrogen peroxide. 1071 00:52:14,580 --> 00:52:16,910 So you don't see any dimedone derivative, 1072 00:52:16,910 --> 00:52:20,280 and then you increase the concentration. 1073 00:52:20,280 --> 00:52:22,760 But you get to the highest concentration 1074 00:52:22,760 --> 00:52:25,730 here that they looked at. 1075 00:52:25,730 --> 00:52:29,500 So it's 100 micromolar versus 50 micromolar 1076 00:52:29,500 --> 00:52:32,230 in the protein they used. 1077 00:52:32,230 --> 00:52:34,330 But what did this immediately tell you? 1078 00:52:34,330 --> 00:52:39,310 Did any of you look at this data very carefully? 1079 00:52:39,310 --> 00:52:40,480 What is this? 1080 00:52:40,480 --> 00:52:46,290 This guy here is associated with a [INAUDIBLE] group that 1081 00:52:46,290 --> 00:52:49,260 is only reacted with iododimedone, 1082 00:52:49,260 --> 00:52:51,780 so if you got 100% yield, what does that tell you? 1083 00:52:51,780 --> 00:52:54,840 This tells you the maximum amount of material 1084 00:52:54,840 --> 00:52:56,140 you're going to observe. 1085 00:52:56,140 --> 00:52:59,130 So if you look at this peak, and you look at that peak, 1086 00:52:59,130 --> 00:53:00,510 you can't do this by eyeball. 1087 00:53:00,510 --> 00:53:02,730 You need to do this quantitatively. 1088 00:53:02,730 --> 00:53:04,530 The phosphor images or methods that 1089 00:53:04,530 --> 00:53:06,234 allow us to do this quantitatively. 1090 00:53:06,234 --> 00:53:06,900 What do you see? 1091 00:53:06,900 --> 00:53:08,402 AUDIENCE: It's not at the max. 1092 00:53:08,402 --> 00:53:10,110 JOANNE STUBBE: Yeah, it's not at the max. 1093 00:53:10,110 --> 00:53:12,510 And so what we'll do next time-- 1094 00:53:12,510 --> 00:53:15,690 so these are sort of controls, and the question 1095 00:53:15,690 --> 00:53:18,960 is how effective is this reagent, 1096 00:53:18,960 --> 00:53:24,330 and if you start hanging stuff off of your dimedone over here, 1097 00:53:24,330 --> 00:53:26,970 are you going to change the rate of modification? 1098 00:53:26,970 --> 00:53:30,150 Can it get into the active site where this SOH actually 1099 00:53:30,150 --> 00:53:31,560 is, these are the kinds of things 1100 00:53:31,560 --> 00:53:35,250 we're going to talk about next time when we look a little bit 1101 00:53:35,250 --> 00:53:38,082 more at the details of the reaction with this, 1102 00:53:38,082 --> 00:53:39,540 and you should look at the reaction 1103 00:53:39,540 --> 00:53:44,790 with gap dehydrogenase, which is another control enzyme they 1104 00:53:44,790 --> 00:53:47,019 ended up looking at it, because what they do 1105 00:53:47,019 --> 00:53:49,560 is address what the issues are that you're going to encounter 1106 00:53:49,560 --> 00:53:52,030 when you get into something real that you care about. 1107 00:53:52,030 --> 00:53:54,120 And that's much more complicated. 1108 00:53:54,120 --> 00:53:57,320 OK so that's it.