Mass spectra of peptides and proteins - and LC analysis of proteomes Stephen Barnes, PhD 4-7117 sbarnes@uab.edu
Overview A mass spectrum Electrospray MS Analysis of intact proteins Molecular weight calculations Max Entropy Peptides Purity Integration of MS with LC and CE Multidimensional LC Tandem MS Identifying modification sites
A mass spectrum of several peptides from a tryptic digest 1366.82 2452.17 841.3 9 8 7 % Intensity 6 5 4 3 1494.9 2 1 1267.76 2164.5 2646.33 2517.29 1388.78 2186.2 1544.76 3141.45 3163.46 9 152 214 276 338 4 Mass (m/z)
Isotope profile of individual peptide ion 1366.82 2452.17 841.3 8 % Intensity 6 4 2 9 152 214 276 338 4 Mass (m/z) % Intensity 8 6 4 1366.82 1367.82 1368.82 2 1369.83 1 1346. 1354.4 1362.8 1371.2 1379.6 1388. Mass (m/z)
How to represent the mass of compound? At low mass resolution (where the isotope peaks cannot be resolved) what is observed is the average mass At high resolution where the isotopic peaks are fully resolved, then we can determine the monoisotopic mass for each one
Take home question (due Feb 6th) 1. What is the monoisotopic mass of human cytochrome C? Hint: workout the empirical formula of hcyt C 2. What is the molecular weight of the most abundant species of human Cyt C? Hint: assume that the abundance of 13 C is 1.% of total carbon atoms - do not worry about 2 H or other isotopes
ESI-MS and purity of peptides Guarantees of purity based on observation of a single peak by reverse-phase HPLC and by it gave the correct sequence when analyzed by Edman degradation are hollow.the lower spectrum was of a pure HPLC peak. The method of purification was amended and the upper spectrum was obtained
Ionizing proteins and peptides + H 3 NCHR 1 CO(NHCHR n CO) n NHCHR 2 COOH is the ion that s found in dilute acid solution If there are internal basic residues, then the ions will be of the form [M+nH] n+, where n = 1, 2, etc. A tryptic peptide will have a N-terminal amino group and an amino group from Arg or Lys If the peptide has a mol. wt. of Da, then the singly charged ion will have a m/z of 1, whereas the doubly charged ion has a m/z of 51
ESI mass spectrum of ribonuclease Cumulative MW estimate = 13,68.29 SD = 2.94 Peak (m/z) Intensity Charge (est.) Mol. Wt. (Est.) 978. 7,778 14. 13,677.89 1,53. 18,532 13.2656 13,675.9 1,141. 59,87 11.95446 13,679.91 1,245. 33,275 1.96146 13,683.91 1,369. 32,39 1.3219 13,679.92 1,521. 35,668 8.99995 13,679.93 1,711. 16,624 7.99996 13,679.94 1,956. 3,333 6.97955 13,684.94
Calculation of molecular weights and ion states For two ions in a series for a peptide of molecular weight M, the lower m/z value (x) will be for the n+1 ion state and the larger m/z value (y) will be for the n+ ion state. - (1) (M+n)/n = y - (2) (M+n+1)/(n+1) = x Hence - (3) M+n = ny and M = ny-n - (4) M+n+1 = (n+1)x and M = (n+1)x-(n+1) Hence - ny-n = (n+1)x - (n+1) - ny-n-xn+n = x-1 - n(y-x) = x-1 - n = (x-1)/(y-x) The value of n can then be substituted in equation (1) to obtain the molecular weight of the peptide
Oxidized Aprotinin ESI mass spectrum % 816.759 933.433 188.845 483 Control Aprotinin ESI mass spectrum % 814.836 931.12 186.19 553 112.455 8 9 1 12 13 m/z Deconvoluted mass spectra Oxidized % 6527 6543 6511 651 6.21e3 1.21e3 Control % Junlong Shao 64 65 66 67 mass
Deconvolution of oxidized forms of β-lactoglobulin 1225.144 1312.467 136 1148.588 1413.476 % 122.515 181.69 153.979 1 12 13 14 15 16 17 m/z 18362 1.39e3 % 18329 18345 18426 18378 18393 18458 18489 1855 18522 1854 18575 183 184 185 186 mass Junlong Shao
LC/MS of 4HNE-Modified Cytochrome C 12356 Native Cytochrome C 12512 Cytochrome C + One 4HNE Cytochrome Michael Addition (+156) C+One 4HNE Schiff Base (+138) % 12494 Cytochrome C + Two 4HNE Michael Addition (+312) 12668 Cytochrome C+Three 4HNE Michael Addition (+467) 12823 123 124 125 126 127 128 Mass Courtesy of Amanda Foxwell
ESI spectrum of bacterially expressed protein A52 111.1 A56/B49 122.47 A48/B42 1192.7 TOF MS ES+ 552 A: 57195.23±12.89 B: 511.25±18.34 C: 15157.6±.94 % C18 843.12 Each ion is [M+nH] n+ A45 1272.8 A41 1396.16 For 5+ charge state of a 5 kda protein, m/z = [5, + 5]/5 = 1,1 7 8 9 1 12 13 14 15 16 17 18 19 2 m/z Courtesy of Mindan Sfakianos
Deconvolution of MS data When several proteins are present, then their multiply charged ion clusters overlap Can this be overcome? - yes, use the MaxEntropy program provided by Micromass
MaxEnt deconvolution of MWs 57,195.2 + 12.9 Da E. coli GRoEL % 5,11.3 + 18.3 Da 6xHis-tag BAT 15,157.6 +.9 Da mass 15 2 25 3 35 4 45 5 55 6 65 Courtesy of Mindan Sfakianos
Summary of determining MW by ESI The multiple charge states of a protein allow: Mol Wt of large proteins to be estimated accurate estimation of mol wt (super SDS- PAGE gel) Important to remember that the protein sample must be free of salt Typically, a sample is cleaned up on a short reverse-phase column prior to electrospray Alternative, use ammonium acetate as buffer
Use of FT-MS in ESI of proteins The very high resolving power of FT-MS enables a direct measure of charge state of an individual ion since each peptide or polypeptide will have several/many isotope peaks The distance in Da between successive isotope peaks of a multiply charged ion is the reciprocal of the number of charges
Bovine Serum Albumin (66 kda) 4.7 T Actively Shielded Magnet ESI: BSA 44+ 44+ 151.6 151.8 1511. m/z 14 16 18 m/z Bruker Daltonics
Chemically modifying an antibody Scheme 1 PTX-2'-OH + Succinic Anhydride PTX-2'-O 2 CCH 2 CH 2 CO 2 H PTX-SX Scheme 2 NHS/EEDQ NH + PTX-SX 2 DMF NH-PTX MAb PTXMAb Ahmad Safavy
Structure of modified antibody Ahmad Safavy
Modification of an antibody by MALDI-TOF 9 8 BSA Unreacted Ab B 152223 917.6 7 6 A 76337 % Intensity 5 4 3 [M+2H] 2+ [M+H] + 2 1 6. 87975.4 11595.8 143926.2 17191.6 199877. Mass (m/z) Reacted Ab C 155148 67.1 9 8 7 6 BSA D 77246 [M+2H] 2+ Δ = 2925 Da % Intensity 5 4 3 [M+H] + 2 1 6 87982 115964 143946 171928 19991 Mass (m/z) Ahmad Safavy
Example of MS to detect a amino acid modification Cytochrome c, a mitochondrial enzyme, was reacted with 4-hydroxynonenal, an aldehyde formed by oxidation of long chain, unsaturated fatty acids Site of attachment believed to be on lysine groups (to form a Schiff s base) However, increase in MW consistent with Michael addition Protein hydrolyzed with trypsin
Cytochrome C Modified by HNE MALDI-TOF Mass Spectrum [M+H] 12365.22 CONTROL + 12521.8 1268.12 PROTEIN + ONE 4HNE - PROTEIN + TWO 4HNE % Intensity 12837.86 PROTEIN +THREE 4HNE [M+2H] 2+ 626.97 [2M+H] + 2527.3 88 166 244 322 4 Mass (m/z) MALDI spectra usually contain only the molecular ion [M+H] +. This is an advantage since it maximizes the signal, but is a disadvantage in that it gives no clues as to structure.
Triple quad versus Q-tof and sensitivity Gas Sample solution 5 KV - - - + - - + - - - N2 Collision gas Q1 Q2 Q3 Detector Q1 Collision gas Q2 Electrostatic reflector The quadrupole analyzer (Q3) is slow and insensitive - it s a filter - thus throws away large amounts of data TOF detector TOF detector collects all ions generated and yields fmol rather than pmol sensitivity Also gives far greater mass accuracy - from ppm on the triple quad to <2 ppm on the Q-tof Crucially important for automated interpretation of MS-MS spectra to yield amino acid sequence
Tandem mass spectrometry on a triple quadrupole instrument Gas Sample solution 5 KV - - - + - - + - - - N2 Collision gas Q1 Q2 Q3 Detector Daughter ion spectra Parent ion spectra Multiple reaction ion scanning
Daughter ion tandem MS Gas Sample solution 5 KV - - - + - - + -- - - N2 Collision gas Q1 Q2 Q3 Detector The molecular ion is selected in Q1, collided with Argon gas in Q2, and daughters analyzed in Q3
Parent ion tandem MS Gas Sample solution 5 KV - - - + - - + - - - N2 Collision gas Q1 Q2 Q3 Detector All molecular ions allowed into Q1, collided in Q2, and a selected daughter ion measured in Q3. This could be an O-GlcNAc fragment (m/z 24). The parent peptide ions containing an O-GlcNAc will be revealed. Very difficult to do this experiment on a Q-tof.
Multiple reaction ion monitoring (MRM) Gas Sample solution 5 KV - - - + - - + - - - N2 Collision gas Q1 Q2 Q3 Detector A single molecular ion selected in Q1, collided in Q2 and a selected daughter ion measured in Q3. Up to 8 pairs of parent/daughter ions can be measured. This is a quantitative analysis. Can t be done on a Q-tof.
MRM analysis of isoflavones in dog plasma 241 /119 18,115 Rel. Int. (%) Rel. Int. (%) 5 5 253 /223 269 /133 Equol Daidzein 875 19 The combination of parent ion and daughter ion allows specific detection of each of these isoflavonoids, even when they are not chromatographically separable Rel. Int. (%) 5 269 /149 Genistein 3565 The figure in the right hand corner is the full scale intensity for each channel Rel. Int. (%) 5. 1 1. 35 2. 68 3. 12 Apigenin 4. 5. 135 169 6. 23 Apigenin is an internal standard and is an isomer of genistein Time (min)/scan
LC-MS of peptide mixtures waste pre-column for desalting Load sample Analytical reverse phase column 75 µm i.d. x 15 cm Flow rate 2 nl/min Acetonitrile gradient Collision gas TOF detector Q1 Q2 Electrostatic reflector
Qtof Tryptic Digest of Control Peptide 12 159 256 37 483 62 677 79 937 994 115 T G P N L H G L F G R 1168 167 11 913 799 686 549 492 379 232 175 % Y 6 686.36 Y 9 11.53 Y 5 549.31 b 6 62.31 Y 7 799.46 Y 8 913.47 Y 1 167.53 2 3 4 5 6 7 8 9 1 12 Amanda Foxwell
Qtof Tryptic Digest of Modified Peptide 12 159 256 37 483 776 833 946 193 115 136 T G P N L H G L F G R 1324 1223 1166 169 955 842 549 492 379 232 175 1168.52 % b 5 483.23 139.1 Y 5-549.26 Y 1 175.1 Y 3 Y 6 842.44 379. 776. Y 7 955. b 6 Y 9 1166.56 2 3 4 5 6 7 8 9 1 12 13 Mass Amanda Foxwell
Conclusions of experiment A peptide was identified that increased its molecular weight by 156 Da Tandem MS revealed that the 4-HNE was attached to His-33 to form a Michael adduct
MUDPIT - MUltilti-Dimensionalimensional Protein Identification Technology NH 4 Cl (1-2 mm step gradient) Cation exchange column (H + ) 1 mm 2 mm -4% MeCN gradient Hydrolyze everything! For a cell expressing 5, proteins, this leads to >, peptides 4 mm 6 mm 8 mm mm 2 mm nanolc Can be fractionated, but still 1,-2, to differentiate Enormous bioinformatics problem MS-MS analysis on Qqtof Massive computing John Yates
Connecting CE and LC to MALDI analysis CE analysis nanolc analysis Creates 2 mm wide tracks that can be scanned by MALDI laser for MS analysis Parallel capture of effluents of 8 nanolc separations on Mylar - can be scanned simultaneously by fast laser
Pros/Cons of laying down LC or EC separations on matrix plate Allows off-line analysis both in real time and then in a retrospective mode MALDI-TOF analysis is very fast Can do TOF-TOF MS-MS analysis BUT what happens chemically on the acidic environment on the surface of the plate during storage? Also, can the laser beam cause chemical changes?