Comparison of mass spectrometers performances Instrument Mass Mass Sensitivity resolution accuracy Quadrupole 1 x 10 3 0.1 Da* 0.5-1.0 pmol DE-MALDI 2 x 10 4 20 ppm 1-10 fmol peptide 1-5 pmol protein Ion trap 1 x 10 3 0.1 Da* 10-20 fmol FT-ICR 1 x 10 6 <1 ppm 20 amole *depends on the mass window being used
Mass spectra of peptides and proteins - and LC analysis of proteomes Stephen Barnes, PhD
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 MALDI spectra Tryptic fingerprinting
A mass spectrum of several peptides from a tryptic digest 100 1366.82 2452.17 8401.3 90 80 70 % Intensity 60 50 40 30 1494.90 20 10 0 1267.76 2164.05 2646.33 2517.29 1388.78 2186.02 1544.76 3141.45 3163.46 900 1520 2140 2760 3380 4000 Mass (m/z) 0
Isotope profile of individual peptide ion 100 1366.82 2452.17 8401.3 80 % Intensity 60 40 20 0 900 1520 2140 2760 3380 0 4000 Mass (m/z) % Intensity 100 80 60 40 1366.82 1367.82 1368.82 20 1369.83 1 1346.0 0 1354.4 1362.8 1371.2 1379.6 1388.0 0 Mass (m/z)
ESI spectrum of bacterially expressed protein A52 1101.1 100 A56/B49 1022.47 A48/B42 1192.70 TOF MS ES+ 552 A: 57195.23±12.89 B: 50011.25±18.34 C: 15157.60±0.94 % C18 843.12 Each ion is [M+nH] n+ A45 1272.08 A41 1396.16 For 50+ charge state of a 50 kda protein, m/z = [50,000 + 50]/50 = 1,001 700 0 80 0 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z Courtesy of Mindan Sfakianos
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 1000 Da, then the singly charged ion will have a m/z of 1001, whereas the doubly charged ion has a m/z of 501
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
ESI mass spectrum of ribonuclease Cumulative MW estimate = 13,680.29 SD = 2.94 Peak (m/z) Intensity Charge (est.) Mol. Wt. (Est.) 978.00 7,778 14.00000 13,677.89 1,053.00 18,532 13.02656 13,675.90 1,141.00 59,087 11.95446 13,679.91 1,245.00 33,275 10.96146 13,683.91 1,369.00 32,390 10.03219 13,679.92 1,521.00 35,668 8.99995 13,679.93 1,711.00 16,624 7.99996 13,679.94 1,956.00 3,333 6.97955 13,684.94
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 100 57,195.2 + 12.9 Da E. coli GRoEL % 50,011.3 + 18.3 Da 6xHis-tag BAT 15,157.6 + 0.9 Da 0 mass 15000 20000 25000 30000 35000 40000 45000 50000 55000 60000 65000 Courtesy of Mindan Sfakianos
LC/MS of 4HNE-Modified Cytochrome C 100 % 0 12356 Native Cytochrome C 12494 Cytochrome C+One 4HNE Schiff Base (+138) 12512 Cytochrome C + One 4HNE Michael Addition (+156) 12668 12300 12400 12500 12600 12700 12800 Mass Cytochrome C + Two 4HNE Michael Addition (+312) 12823 Cytochrome C+Three 4HNE Michael Addition (+467) Courtesy of Amanda Foxwell
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+ 1510.6 1510.8 1511.0 m/z 1400 1600 1800 m/z Bruker Daltonics
ESI-MS and purity of peptides Guarantees of purity based on observation of a single peak by reversed-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
LC-MS of peptide mixtures waste pre-column for desalting Load sample Analytical reverse phase column 75 mm i.d. x 15 cm Flow rate 200 nl/min Acetonitrile gradient Collision gas TOF detector Q1 Q2 Electrostatic reflector
Tandem mass spectrometry on a triple quadrupole instrument Daughter ion spectra The molecular ion is selected in Q1, collided with Ar gas in Q2, and daughters analyzed in Q3 Parent ion spectra All molecular ions allowed into Q1, collided in Q2, and a selected daughter ion measured in Q3 Multiple reaction ion monitoring (MRM) 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
MUDPIT - MUlti lti-dimensionalimensional Protein Identification Technology NH 4 Cl (1-200 mm step gradient) Cation exchange column (H + ) 10 mm 20 mm 0-40% MeCN gradient Hydrolyze everything! For a cell expressing 5,000 proteins, this leads to >100,000 peptides 40 mm 60 mm 80 mm 100 mm 200 mm nanolc Can be fractionated, but still 10,000-20,000 to differentiate Enormous bioinformatics problem MS-MS analysis on Qqtof Massive computing John Yates
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?
Example of MS-MS to detect a 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
Qtof Tryptic Digest of Control Peptide 100 102 159 256 370 483 620 677 790 937 994 1150 T G P N L H G L F G R 1168 1067 1010 913 799 686 549 492 379 232 175 % Y 6-686.357 Y 9-1010.536 Y 5-549.308 Y 7-799.462 Y 10-1067.535 B 6-620.306 Y 8-913.470 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Amanda Foxwell
Qtof Tryptic Digest of Modified Peptide 100 102 159 256 370 483 776 833 946 1093 1150 1306 T G P N L H G L F G R 1324 1223 1166 1069 955 842 549 492 379 232 175 1168.520 % B 5-483.225 Y 6-842.437 139.104 Y 5-549.266 Y 9-1166.563 Y 3-379.001 B 6-776.001 Y 1-175.101 Y 7-955.001 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 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
MALDI-TOF MS Whole proteins Tryptic fingerprinting MS-MS
Cytochrome C Modified by HNE MALDI-TOF Mass Spectrum 100 [M+H] 12365.22 CONTROL + 12521.80 12680.12 PROTEIN + ONE 4HNE - PROTEIN + TWO 4HNE % Intensity 12837.86 PROTEIN +THREE 4HNE [M+2H] 2+ 6260.97 [2M+H] + 25207.30 1000 8800 16600 24400 32200 40000 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.
Protein analysis by MALDI 2003 destain Eppendorf tube Speed-Vac Water Bath 37 o C Incubate overnight trypsin 1:20 Desalting Ziptip MALDI plate Counts 15000 10000 5000 684.11 789.57 1048.72 1122.79 1165.80 1277.83 1461.00 1705.16 1838.26 1937.22 1994.24 2137.21 2284.25 2384.27 2647.50 0 1000 1500 2000 2500 3000 Mass (m/z)
100 1181.59 90 80 70 % Intensity 60 50 40 1461.75 1443.76 1950.89 30 20 10 1583.79 2368.13 1016.55 2164.05 1034.54 1687.98 1184.60 1756.86 2185.02 2813.32 0 900 1520.6 2141.2 2761.8 3382.4 4003.0 Mass (m/z)
MASCOT Search 1016.55 1034.54 1181.59 1461.75 1443.76 1687.98 1950.80 2368.13 2813.32 Trypsin Digest of porin-p1; Voltage-dependent anion-selective channel
Connecting CE and LC to MALDI analysis CE analysis nanolc analysis A Creates 20 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
Post-source decay experiments in a TOF-mass spectrometer Accelerating pulse Daughter fragment ions formed in the drift region are separated by the reflector. Suitable resolution only occurs over a limited range of m/z values. This can be overcome by recording individual spectra over a wide range of voltage settings (10-12) for the reflector. Alternatively, a curved applied voltage can be used to obtain the daughter ion spectrum in a single experiment. Reflectron
TOF-TOF - high speed MSMS ion generation
Decomposition of -NO 2 group in MALDI-TOF MS Counts 5000 10000 15000 A 927.94 BSA YLYEIAR 1194.77 1250.15 1285.25 1440.40 1480.40 0 800 900 1000 1100 1200 1300 1400 1500 Counts 1000 2000 3000 4000 B 927.3937 972.52 NO2 BSA + TNM 1194.77 1249.2963 1441.61 1480.50 Counts 0 1000 2000 3000 4000 800 900 1000 1100 1200 1300 1400 1500 C 943.1852 BSA + TNM +Dith NH2 1194.4332 1251.12 1283.67 1441.61 0 800 900 1000 1100 1200 1300 1400 1500 m/z