Mass Spectrometry on the Cinco de Mayo

Mass Spectrometry on the Cinco de Mayo Peter Lobel, Ph.D. Resident Member, Center for Advanced Biotechnology and Medicine Professor of Biochemistry and Molecular Biology, Rutgers-RWJMS Director, Rutgers-RWJMS Biological Mass Spectrometry Facility Member, Center for Integrative Proteomics Research lobel@cabm.rutgers.edu Images courtesy of David Fenyö, Nathan Yates, Wilhelm Haas, Michael McCoss, AB-SCIEX, Thermo

Outline of today s talk Overview - mass spectrometry in proteomics How mass spectrometers work Looking at spectra - mass and isotopes Quantitative proteomics

intensity What does a mass spectrometer do? Measures mass/charge (m/z) of intact ions and fragments vacuum Ion Source Mass Analyzer Detector Quantitative information m/z Structural information

Use of mass spectrometry in proteomics research protein identification protein processing: proteolytic post-translational modifications (100 s) quaternary structure complex formation steady-state levels turnover rates structural studies (H/D exchange) comparison of samples (disease & controls)

Mass spectrometry is an enabling technology* in the field of proteomics Peptide Sequencing Material required Edman degradation ~pmol (6 x 10 11 molecules) Mass spectrometry fmol 6 x 10 8 molecules Time (data collection) ~10 hours 10 msec Wikipedia *An enabling technology is an invention or innovation, that can be applied to drive radical change in the capabilities of a user or culture. Enabling technologies are characterized by rapid development of subsequent derivative technologies, often in diverse fields. Equipment and/or methodology that, alone or in combination with associated technologies, provides the means to increase performance and capabilities of the user, product or process.

The mass of a single peptide provides sequence constraints but is not sufficient for identification MS 1. MS1 (MS) spectra measure intact peptide ions Relative Abundance 300 400 500 600 700 800 1000 900 1200 1400 1600 1800 m/z Predicted tryptic peptides in database 507.2962 K.RLNIVQDR.F 507.3031 K.ANELLINVK.Y 507.3031 K.IIAIDINNK.K 507.3031 R.VLNLPSVGSK.S 507.3088 R.LNVLSNVVR.K 507.3088 K.SPKSNKKPK.R 507.3213 K.AIILGAQSIK.C... 507.303 Wilhelm Haas observed m/z, z=2 The precursor mass is not enough information for ID

Collision-Induced Dissociation (CID) + + Kinetic energy of parent ions is increased Parent ions undergo energy converting collisions Parent ions fall apart into product ions and neutrals Also referred to collision-activated dissociation (CAD) Nathan Yates

Typical CID fragmentation pattern of peptides H +

Combined residue mass for two amino acids Gly Ala Ser Pro Val Thr Cys Lxx Asn Asp Gln Lys Glu Met His Phe Arg Cmc Tyr Trp AA 57 71 87 97 99 101 103 113 114 115 128 128 129 131 137 147 156 161 163 186 Gly 57 114 Ala 71 128 142 Ser 87 144 158 174 Pro 97 154 168 184 194 Val 99 156 170 186 196 198 Thr 101 158 172 188 198 200 202 Cys 103 160 174 190 200 202 204 206 Lxx 113 170 184 200 210 212 214 216 226 Asn 114 171 185 201 211 213 215 217 227 228 Asp 115 172 186 202 212 214 216 218 228 229 230 Gln 128 185 199 215 225 227 229 231 241 242 243 256 Lys 128 185 199 215 225 227 229 231 241 242 243 256 256 Glu 129 186 200 216 226 228 230 232 242 243 244 257 257 258 Met 131 188 202 218 228 230 232 234 244 245 246 259 259 260 262 His 137 194 208 224 234 236 238 240 250 251 252 265 265 266 268 274 Phe 147 204 218 234 244 246 248 250 260 261 262 275 275 276 278 284 294 Arg 156 213 227 243 253 255 257 259 269 270 271 284 284 285 287 293 303 312 Cmc 161 218 232 248 258 260 262 264 274 275 276 289 289 290 292 298 308 317 322 Tyr 163 220 234 250 260 262 264 266 276 277 278 291 291 292 294 300 310 319 324 326 Trp 186 243 257 273 283 285 287 289 299 300 301 314 314 315 317 323 333 342 347 349 372 Nathan Yates

Sequencing a peptide ion using tandem MS (MS/MS) 1. MS MS/MS Relative Abundance 507 2. 4. Isolate Fragment one peptide ions are measured in MS/MS 3. Collide with inert gas (CID) to fragment Relative Abundance 300 400 500 600 700 800 1000 900 1200 1400 1600 1800 m/z 200 400 600 800 1000 1200 m/z 507.303 observed m/z Wilhelm Haas MS/MS fragments provide information about the amino acid sequence for manual interpretation or database searching

Sequencing a peptide ion using tandem MS (MS/MS) Wilhelm Haas H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib nd_1884 #4848 RT: 28.00 AV: 1 NL: 5.36E1 T: ITMS + c NSI d Full ms2 459.76@cid35.00 [115.00-930.00] 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 453.5731 583.5828 370.4370 521.4109 624.7378 568.1208 639.3729 308.8508 181.3454 267.4216 755.6002 147.0887 667.3040 680.5607 823.1918 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z Mass Spec Database Relative Abundance Experimental Spectra H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib nd_1884 #4848 RT: 28.00 AV: 1 NL: 5.36E1 H:\bin\nd_1884 T: ITMS + c NSI d Full ms2 459.76@cid35.00 [115.00-930.00] 4/9/2007 8:34:29 PM pep lib 453.5731 100 H:\bin\nd_1884 nd_1884 #4848 RT: 28.00 AV: 1 NL: 5.36E1 4/9/2007 8:34:29 PM pep lib T: ITMS + c NSI d Full 95 ms2 459.76@cid35.00 [115.00-930.00] 453.5731 H:\bin\nd_1884 nd_1884 #4848100RT: 28.00 90 AV: 1 NL: 5.36E1 4/9/2007 8:34:29 PM pep lib T: ITMS + c NSI d Full ms2 459.76@cid35.00 [115.00-930.00] 95 85 453.5731 nd_1884 #4848 RT: 100 28.00 AV: 1 NL: 5.36E1 80 T: ITMS + c NSI d Full ms2 90 95 459.76@cid35.00 [115.00-930.00] 75 453.5731 100 85 90 70 95 80 85 75 65 90 80 60 70 85 75 55 65 80 70 50 60 246.3161 75 65 45 441.1335 583.5828 55 40 70 60 50 35 65 55 45 30 583.5828 246.3161 441.1335 60 50 40 25 45 370.4370 521.4109 55 35 583.5828 20 624.7378 568.1208 40 50 30 15 639.3729 246.3161 441.1335 35 308.8508 25 10 181.3454 267.4216 755.6002 45 521.4109 147.0887 370.4370 583.5828 667.3040 30 20 5246.3161 624.7378 568.1208 823.1918 40 441.1335 680.5607 25 15 0 639.3729 35 150 200 308.8508 250 370.4370 521.4109 300 350 400 450 500 550 600 650 700 750 800 850 900 20 10 181.3454 624.7378 267.4216 568.1208 m/z 755.6002 30 147.0887 667.3040 15 246.3161 639.3729 5 441.1335 823.1918 308.8508 680.5607 25 10 0 181.3454 267.4216 755.6002 370.4370 521.4109 147.0887 150 200 250 300 350 400 450 500 550 600 667.3040 20 624.7378 650 700 750 800 850 900 5 568.1208 823.1918 m/z 680.5607 15 639.3729 0 150 200308.8508 250 300 350 400 450 500 550 600 650 700 750 800 850 900 10 181.3454 267.4216 755.6002 m/z 147.0887 667.3040 5 823.1918 680.5607 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z Precursor & fragment ions Peptide ID Relative Abundance Relative Abundance Relative Abundance Relative Abundance Search Engine Theoretical Spectra m/z All rely on the database peptide sequences (largely predicted from DNA and RNA sequences)

base peak intensity 80 H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib 75 nd_1884 #4848 RT: 28.00 AV: 1 NL: 5.36E1 T: ITMS + c NSI d Full ms2 70459.76@cid35.00 [115.00-930.00] 453.5731 100 65 95 60 90 H:\bin\nd_1884 55 4/9/2007 8:34:29 PM pep lib 85 50 nd_1884 #4848 RT: 80 28.00 AV: 1 NL: 5.36E1 T: ITMS + c NSI d Full ms2 459.76@cid35.00 [115.00-930.00] 453.5731 100 75 45 95 70 40 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 65 H:\bin\nd_1884 4/9/2007 8:34:29 PM pep lib nd_1884 #4848 RT: 28.00 AV: 1 NL: 5.36E1 T: ITMS + c NSI d Full ms2 459.76@cid35.00 [115.00-930.00] 100 95 90 85 35 453.5731 60 30 246.3161 441.1335 55 25 50 370.4370 521.4109 20 624.7378 45 568.1208 583.5828 15 639.3729 40 308.8508 35 10 181.3454 267.4216 755.6002 147.0887 667.3040 30 5 246.3161 823.1918 441.1335 680.5607 25 0 370.4370 583.5828 521.4109 20 150 200 250 300 350 400 450624.7378 500 550 600 650 700 750 800 850 900 568.1208 15 639.3729 m/z 308.8508 10 181.3454 267.4216 755.6002 246.3161 441.1335 147.0887 667.3040 5 823.1918 370.4370 521.4109 680.5607 0 624.7378 568.1208 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 639.3729 m/z 308.8508 181.3454 267.4216 755.6002 147.0887 667.3040 823.1918 680.5607 583.5828 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z Tryptic digest A typical proteomics experiment Mass Spectrometer HPLC Pumps Column LC-MS/MS Separate microgram quantities of peptides on a capillary C18 column Relative Abundance Relative Abundance Relative Abundance Data Analysis Wilhelm Haas Column I.D. = 75 μm time organic concentration in mobile phase We can identify 10,000 peptides in a 90 min run

How do mass spectrometers work? High Vacuum System Inlet Ion source Mass Analyzer Detector Data System

Mass analyzers use forces to manipulate ions dv F ma m z( E v B) dt m dv E v B z dt

detector Time-of-flight (TOF) mass analyzer Electric potential Drift region (flight tube) + + + + 2000 V d Ions are formed in pulses. The drift region is field free. Measures the time for ions to reach the detector. Small ions reach the detector before large ones.

detector Time-of-flight (TOF) mass analyzer Electric potential Drift region (flight tube) + + + + 2000 V d Energy uptake is E el =qv=ezv where e=charge/electron Conversion of potential energy to kinetic energy. ezv = ½ mv 2 The drift region is field free. Known distance, d. Measures the time for ions to reach the detector. v=d/t m/z=2evv 2 =2eVd 2 /t 2

Quadrupole mass analyzer Oscillating electric fields, operates as a mass filter. Has four parallel metal rods. Lets one mass pass through at a time. Can scan through all masses or sit at one fixed mass.

Quadrupoles have variable ion transmission modes m2 m4 m1 m3 m4 m3 m2 m1 mass scanning mode (let different m/z through as function of time, collect mass spectrum) m2 m4 m1 m3 m2 m2 m2 m2 single mass transmission mode let single m/z through, measure intensity

Triple quadrupole MS (tandem in space)

Selected Reaction Monitoring (SRM) on a triple quadrupole mass spectrometer Select Fragment Select precursor ion in Q1 precursor ion in Q2 product ion in Q3 (Collision cell containing inert gas)

Ion Trap Mass Spectrometer

Ion Trap MS process transfer and focus ions accumulate ions compress ion cloud in center apply resonance voltage to eject ions selectively

MS/MS on an ion trap mass spectrometer Tandem-in-Time (allows MSn) Eject < M Eject > M Daughter Ion Scanning rf CAD Ionization Precursor Ion Isolation Collisionally Activated Dissociation Product Ion Scanning 0 Injection Time (ms)

How do mass spectrometers work? High Vacuum System Inlet Ion source Mass Analyzer Detector Data System

Matrix Assisted Laser Desorption/Ionization (MALDI) Sample plate hn Laser 1. Sample is mixed with matrix (X) and dried on plate. 2. Laser flash ionizes matrix molecules. MH + 3. Sample molecules (M) are ionized by proton transfer: XH + + M MH + + X. +/- 20 kv vacuum Grid (0 V) Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) MH+ 225, 207 Frequently used MALDI Matrix for proteins, large peptides

Electrospray Ionization (ESI) Frequently used with liquid chromatography (LC-MS) Pressure = 1 atm Inner tube diam. = 100 um Sample Inlet Nozzle (Lower Voltage) Partial vacuum N 2 MH + Sample in solution N 2 gas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + MH 2 +2 MH 3 +3 High voltage applied to metal sheath (~4 kv) Charged droplets

Electrospray Ionization (ESI) Frequently used with liquid chromatography (LC-MS) Electrospray Ionization Protonated Molecules From µlc (M+H) + Nanospray (typically ~0.2 L/min) do not use sheath gas. To MS

Mass spectrometers are frequently named based on their ion source & mass analyzer High Vacuum System Inlet Ion source Mass Analyzer Detector Data System MALDI Electrospray TOF Quadrupole (Q) Ion trap Orbitrap (orbi) Hybrid: TOF-TOF QQQ Q-TOF Q-Orbi Ion trap-orbi

Mass Spectra Mass units Isotopes the good, the bad & the ugly

How is mass defined? Numerical value to the intrinsic property of mass is based in reference to the most abundant isotope of carbon, 12 C (6 protons & 6 neutrons). One unit of mass is defined as a Dalton (Da). A Da is defined as 1/12 the mass of one 12 C atom. Thus, one 12 C atom has a mass of 12.0000 Da.

Most elements have >1 stable isotope For example, most carbon atoms have a mass of 12 Da, but in nature, 1.11% of C atoms have an extra neutron, making their mass 13 Da. Isotope composition of molecules depends on molecular formula and isotope distribution of component atoms (binomial distribution). Element Isotope Abundance Hydrogen 1H 99.985 2H 0.015 Carbon 12C 98.890 13C 1.110 Nitrogen 14N 99.630 15N 0.370 Oxygen 16O 99.759 17O 0.037 18O 0.204

Isotope pattern (C 1 ) +1 Monoisotopic peak, 12 C 1 ~ 99% of total (0.989) 1

Isotope Pattern (C 60 ) +1 Monoisotopic peak, 12 C 60 ~ 51% of total (0.989) 60

Isotope Pattern (C 300 ) +1 Monoisotopic peak, 12 C 300 ~ 4% of total (0.989) 300

Effect of isotope abundance on mass measurements Intensity 1002 1004 1006 1008 m/z Intensity 2004 2006 2008 2010 m/z Intensity Intensity 4014 4016 4018 4020 4022 m/z 0 500 1000 1500 2000 2500 3000 3500 4000 mass/charge (m/z; z=1)

Isotope clusters allow charge state determination Intensity Intensity Intensity 1 1 1 1+ m/z 0.5 0.5 2+ 0.5 m/z 0.33 0.33 3+ 0.33 m/z David Fenyö

Relative Intensity Resolution M R = = resolving power M Resolution = minimum peak separation, M, which allows to distinguish two ion species M = full width at half maximum (FWHM) 500 50 % I 499.5 I 500.0 I 500.5 m/z I 501.5 502.0 I Resolution = M/ M = 500/0.5 = 1000 David Fenyö

Monoisotopic mass Monoisotopic mass corresponds to lowest mass peak When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement. Spacing of adjacent isotope peaks ( m/z, measured) gives z as know m)

Average mass Average mass corresponds to the centroid of the unresolved peak cluster When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the isotope peaks in the cluster, which is the same as the average or chemical mass.

Why we typically analyze peptides instead of intact proteins Protein heterogeneity: proteolytic processing post translational modifications Stability in gas phase Natural isotope abundance Better resolution and accuracy at lower masses

Quantitative proteomics Label-free Stable isotope labeled ( 13 C, 15 N, and/or 18 O) Metabolic labeling Synthetic peptides and proteins Chemical modification

Relative Abundance Quantification of proteins by targeted MS/MS using internal standards NH 2 Protein of Interest COOH Analyte Peptide AQTDIDSPQNLVTDR IS Peptide NH 2 AQTDIDSPQNLVTDR* COOH QconCAT Protein QQQ Detection: Selected Reaction Monitoring provides the high sensitivity and amino acid selectivity needed to detect peptides in complex mixtures Quantification: Peptide concentrations are obtained by multiplying the internal standard concentration by the signal ratio of the analyte / internal standard QQQ 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 18 20 22 24 26 28 30 Time (min) LC - SRM Analyte Signal = 200 Int. Std. Signal = 1000 42

Quantitative analysis using an internal standard Intensity Intensity Sample (m/z = M) Spiked with Internal Standard (m/z = M+i) M M+ i m/z time 1000 Standard (vary) Endogenous peptide (fixed) Peak Area 100 10 1 0.1 1 10 100 1000 10000 100000 amol internal standard

Quantitiatve Proteomics: isobaric labeling reagents Mol. Cell. Proteomics 3, 1154-1169(2004)

% Intensity MS 1 : multiple precursor (peptide) ions mass/charge (m/z) 4 7 0 0 M S/M S Pre c u rs o r 9 5 2.5 6 7 Sp e c # 1 [BP = 1 1 5.1, 7 8 5 4 4 ] 100 115. 1359 7. 9E+4 90 80 70 117. 1397 175. 1362 Man2b1 888 -TQFSGLR- 894 % I nt ensi t y 60 116. 1317 50 70. 0928 40 87. 1082 374. 2191 521. 2928 30 112. 1105 218. 1851 432. 2634 120. 1099 665. 3603 778. 4455 579. 3332 20 71. 0950 145.1292 707. 4360 346.2271 59. 0793 129. 1252 229. 1705 417. 2315 493. 2941 787. 4261 10 228. 1710 328. 2076 516. 2964 590. 3156 30. 0611 130. 1267 414.2555 690.3844 753. 3346 832. 4753 905. 4017 124. 1270 192. 1629 253. 1553 344. 2373 477. 3035 560. 3219 630. 3959 0 9. 0 208. 6 408. 2 607. 8 807. 4 1007. 0 Mass ( m/ z)

114 115 116 117 Expanded scale Reporter ion region 4 7 0 0 M S/M S Pre c u rs o r 9 5 2.5 6 7 Sp e c # 1 [BP = 1 1 5.1, 7 8 5 4 4 ] 100 115. 1359 7. 9E+4 90 80 70 117. 1397 175. 1362 Man2b1 888 -TQFSGLR- 894 % I nt ensi t y 60 116. 1317 50 70. 0928 40 87. 1082 374. 2191 521. 2928 30 112. 1105 218. 1851 432. 2634 120. 1099 665. 3603 778. 4455 579. 3332 20 71. 0950 145.1292 707. 4360 346.2271 59. 0793 129. 1252 229. 1705 417. 2315 493. 2941 787. 4261 10 228. 1710 328. 2076 516. 2964 590. 3156 30. 0611 130. 1267 414.2555 690.3844 753. 3346 832. 4753 905. 4017 124. 1270 192. 1629 253. 1553 344. 2373 477. 3035 560. 3219 630. 3959 0 9. 0 208. 6 408. 2 607. 8 807. 4 1007. 0 Mass ( m/ z)

Parting message Progress in science depends on new techniques, new discoveries, and new ideas, probably in that order Sydney Brenner, 20 March 1980 Biology in the 1980 s, talk at the Friedrich Miescher Institute, Basel, Switzerland