MS-based proteomics to investigate proteins and their modifications
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1 MS-based proteomics to investigate proteins and their modifications Francis Impens VIB Proteomics Core October th 217
2 Overview Mass spectrometry-based proteomics: general workflow Identification of protein post-translational modifications (PTMs) Take-home messages are in red 2
![21239_C1_sn8 #94-983 RT: 29.3-29.17 AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] 1 9 8 7 6 4 4 3 2 1 1 2.3274 2.8292 3.334 4.3398 4.8423 6.289 1.87 3.8319 4.7413.2914 1. 2.416 2.429.8367 6.78 1.](/docs-images/78/77901747/images/3-1.jpg "1. 2. 2. 3. 3. 4. 4... 6. 6. 7. 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [14.-111.] 1 9 8 7 6 4 4 3 2 1 1 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [14.-111.] 1 9 8 7 6 4 4 286.")
![218 399.33 6.4398 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS 479.331 + c NSI d Full ms2 2.33@cid. 92.34 [14.-111.] 12.383 1 9 8 7 43.9283 399.33.377 479.331 92.34 12.383 43.9283 6.4398 772.](/docs-images/78/77901747/images/3-3.jpg "292 818.9.377 479.331 92.34 12.383 64.4697 843.228 6 772.292 7. 187.113 261.16 3 43.9283 371.2819 4 877.878 9.6296 286.218 399.33 146.96 2 4 64.4697 843.228 2 3 4 6 7 8 9 1 11 1 772.292 7. 1 261.")
3 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E T: ITMS + c NSI d Full ms2 2.33@cid. [ ] Mass spectrometry-based proteomics trypsin protein extract peptides LC-MS/MS 1. Liquid Chromatography 2. ElectroSpray Ionization (ESI) 3. Tandem Mass Spectrometry 4. Data analysis peptide masses MS spectra peptide fragment masses spectra algorithm to identify peptide & protein protein database peptide separation C18 nanoflow HPLC peptide ionization (positive charge) cycles of MS and Bio-informatics
4 Protein extraction protein extract Protein extraction: = disrupt the cell wall and cell membrane and bring proteins in solution complete extraction is difficult/impossible strongly dependent on the sample: animal vs. plant vs. prokaryotic cells mechanical cell disruption, e.g. by sonication or by freeze-thawing non-mechanical cell disruption, e.g. by detergents or by chaotropes (e.g. 8 M urea) prevent protein degradation: protease inhibitor cocktails, samples are kept on ice
5 Trypsin digestion trypsin protein extract K/R peptides Trypsin: very specific and highly active protease: cleaves after unmodified Lys or Arg residues generates peptides of about 1 to 2 residues tryptic peptides end on positively charged Lys or Arg, good for ionization
6 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] LC-MS/MS = Liquid Chromatography - Tandem Mass Spectrometry trypsin protein extract peptides LC-MS/MS 1. Liquid Chromatography 2. ElectroSpray Ionization (ESI) 3. Tandem Mass Spectrometry peptide masses MS spectra peptide fragment masses peptide separation C18 nanoflow HPLC peptide ionization (positive charge) cycles of MS and
7 Liquid Chromatography 1. Liquid Chromatography NL: E TIC MS _ q_tc_col _gantasfi_v _input peptide separation C18 nanoflow HPLC Time (min) peptide chromatogram RP-HPLC: Reversed-Phase High Pressure Liquid Chromatography: peptide separation based on hydrophobicity on C18 beads elution by acetonitrile gradient up to +/- % in.1% formic acid, good for ionization nanoflow rates (e.g 3 nanoliter/minute) in capillaries with micrometer diameter (e.g. micron) under very high pressure (up to 1 bar)
8 ElectroSpray Ionisation (ESI) 2. ElectroSpray Ionization (ESI) - aqueous/organic acidified liquid with peptides - small-diameter needle with a high positive voltage - negatively charged voltage on the mass spectrometer inlet Taylor cone peptide ionization (positive charge)
9 ElectroSpray Ionisation (ESI) 2. ElectroSpray Ionization (ESI) - aqueous/organic acidified liquid with peptides - small-diameter needle with a high positive voltage - negatively charged voltage on the mass spectrometer inlet + - sheet gas drying gas (nitrogen) - increased temperature ( C) - vacuum in mass spectrometer Taylor cone peptide ionization (positive charge) ElectroSpray Ionization (ESI) transition from ions in solution to ions in the gas phase + soft ionization method + multiply charged ions - low salt tolerance
10 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] Tandem Mass Spectrometry (MS/MS) 3. Tandem Mass Spectrometry 1 Da 1 peptide masses MS spectra cycles of MS and peptide fragment masses MS spectrum = mass of the intact peptides biomolecules do not have a single mass: peptide envelope
11 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] Tandem Mass Spectrometry (MS/MS) 3. Tandem Mass Spectrometry 1 Da ;y2 peptide masses MS spectra cycles of MS and peptide fragment masses 1 MS spectrum = mass of the intact peptides ;b ;y ;y 387.2;y ;y ;y MS/MS spectrum = mass of the peptide fragments M E P V D P R b-ions b 6 b b 4 y 1 y 2 y 3 y 4 y-ions biomolecules do not have a single mass: peptide envelope isolated peptides are fragmented by collision induced dissociation (CID) mass difference between fragment ions holds sequence information b 3 y b 2 y 6 b 1 1 MS spectrum is followed by a number (e.g. 1), this cycle is repeated constantly random sampling with bias towards most abundant peptides (and thus proteins)
![21239_C1_sn8 #94-983 RT: 29.3-29.17 AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] 1 9 8 7 6 4 4 3 2 1 1 2.3274 2.8292 3.334 4.3398 4.8423 6.289 1.87 3.8319 4.7413.2914 1. 2.416 2.429.8367 6.78 1.](/docs-images/78/77901747/images/12-2.jpg "1. 2. 2. 3. 3. 4. 4... 6. 6. 7. 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [14.-111.] 1 9 8 7 6 4 4 3 2 1 1 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [14.-111.] 1 9 8 7 6 399.")
12 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] Tandem Mass Spectrometry (MS/MS) 3. Tandem Mass Spectrometry 1 Da ;y2 peptide masses MS spectra cycles of MS and peptide fragment masses 1 MS spectrum = mass of the intact peptides ;b ;y ;y 387.2;y ;y ;y MS/MS spectrum = mass of the peptide fragments general undersampling: many unidentified peptides speed to isolate and fragment peptides is a limiting factor hybrid mass spectrometers with multiple mass analyzers such as quadrupole (Q) time of flight (TOF) iontrap orbitrap orbitrap Q-Exactive instrument quadrupole
13 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] Mass spectrometry-based proteomics trypsin protein extract peptides LC-MS/MS 1. Liquid Chromatography 2. ElectroSpray Ionization (ESI) 3. Tandem Mass Spectrometry peptide masses MS spectra peptide fragment masses peptide separation C18 nanoflow HPLC peptide ionization (positive charge) cycles of MS and
14 21239_C1_sn8 # RT: AV: 1 NL: 1.11E6 F: FTMS + p NSI Full ms [3.-2.] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS c NSI d Full ms2 2.33@cid [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E T: ITMS + c NSI d Full ms2 2.33@cid. [ ] Mass spectrometry-based proteomics trypsin protein extract peptides LC-MS/MS 1. Liquid Chromatography 2. ElectroSpray Ionization (ESI) 3. Tandem Mass Spectrometry 4. Data analysis peptide masses MS spectra peptide fragment masses spectra algorithm to identify peptide & protein protein database peptide separation C18 nanoflow HPLC peptide ionization (positive charge) cycles of MS and Bio-informatics
15 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. [ ] _C1_sn #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid [ ] _C1_sn8 #979 RT: 29.1 AV: 1 NL:.E T: ITMS + c NSI d Full ms2 2.33@cid. [ ] Mass spectrometry-based proteomics trypsin protein extract peptides LC-MS/MS Database searching: 4. Data analysis by software algorithms that compare recorded to theoretical spectra generated by in silico digestion of a protein sequence database you can only identify proteins that are in the database popular free algorithm is MaxQuant spectra algorithm to identify peptide & protein protein database Bio-informatics
16 Mass spectrometry can be quantitative vs. sample A isotopic labeling approaches sample B label-free quantification based on non-radioactive, stable isotopes (e.g. 13 C, 18 O, 1 N) metabolic labeling (e.g. SILAC) non-metabolic labeling relies on highly reproducible sample prep & LC almost as accurate as labeling approaches no sample mixing, separate LC-MS/MS sample mixing before LC-MS/MS
17 Overview Mass spectrometry-based proteomics: general workflow Identification of protein post-translational modifications (PTMs) Take-home messages are in red 17
18 Identification of PTMs trypsin protein extract peptides LC-MS/MS Two principles: I. modification leads to a measurable mass difference II. pre-enrichment of modified peptides
19 I. Measurable mass difference: example from the lab Purified protein X mod Question: is the bacterial protein X modified by host enzymes? incubation with host cell nuclear extract MS spectra Acetyl Da Trimethyl min +/- % DLALIKADLAEFEAR? min Protein X gelband Ac + DLALIKADLAEFEAR? +/- % Time (min)
20 I. Measurable mass difference: example from the lab Purified protein X Question: is the bacterial protein X modified by host enzymes? incubation with host cell nuclear extract mod b4; b; b6; 4.42 b7; 7.46 I K A y8;.43 y9; y1: y11; A K I DLALIKADLAEFEAR LntA gelband b 6 b b 4 b 3 b 2 b 1 y 1 y 2 y 3 y 4 y y 6 y8;.43 y9; y1: y11; MS/MS spectra b4; b; b6; b7; 7.46 I K-Ac A A K-Ac I Ac DLALIKADLAEFEAR
21 II. Pre-enrichment of modified peptides trypsin protein extract peptides LC-MS/MS
22 II. Pre-enrichment of modified peptides trypsin protein extract peptides pre-enrichment of modified peptides LC-MS/MS
23 II. Pre-enrichment of modified peptides trypsin protein extract peptides pre-enrichment of modified peptides LC-MS/MS Ubiquitin MQIFVKTLTGKTITLEVEPSDTIENV KAKIQDKEGIPPDQQRLIFAGKQLE DGRTLSDYNIQKESTLHLVLRLRGG trypsin xxxxxxxrxxxxkxxxkxxxxrxxx GG xxxxkxxxk xxx xxxxr xxxxxxxr peptide IP GG xxxxkxxxk LC-MS/MS
24 Acknowledgements An Staes Evy Timmerman Delphi Van Haver Jarne Pauwels Teresa Maia Kris Gevaert Fabien Thery Lia Martina Katie Boucher
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