MS-based proteomics to investigate proteins and their modifications

MS-based proteomics to investigate proteins and their modifications Francis Impens VIB Proteomics Core October th 217

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. 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 772.292 818.9.377 479.331 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. 1 261.16 3 187.113 371.2819 286.218 877.878 9.6296 146.96 2 64.4697 843.228 2 3 1 4 6 7 8 9 1 11 7. 1 187.113 261.16 371.2819 877.878 9.6296 146.96 818.9 6.4398.377 818.9 2 3 4 6 7 8 9 1 11 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 6.4398 21239_C1_sn8 479.331 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. 92.34 [14.-111.] 12.383 1 9 399.33 8 7.377 6.4398 818.9 43.9283 479.331 92.34 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 12.383 T: ITMS + c NSI d Full ms2 2.33@cid. [14.-111.].377 1 43.9283 772.292 4 286.218 399.33 9 479.331 92.34 4 64.4697 843.228 6 12.383 818.9 772.292 8.377 7. 187.113 261.16 3 43.9283 371.2819 286.218 4 7 877.878 9.6296 2 64.4697 146.96 843.228 4 399.33 479.331 92.34 2 1 3 4 6 7 8 9 1 7. 11 6 12.383 1 187.113 261.16 772.292.377 3 371.2819 877.878 9.6296 43.9283 146.96 286.218 2 2 4 3 4 6 7 64.4697 8 9 843.228 399.33 1 11 1 4 7. 1 187.113 261.16 772.292 3 371.2819 877.878 9.6296 146.96 286.218 2 2 3 4 6 7 64.4697 8 9 843.228 1 11 1 7. 1 187.113 261.16 371.2819 877.878 9.6296 146.96 6.4398 818.9 6.4398 818.9 2 3 4 6 7 8 9 1 11 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

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

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

Liquid Chromatography 1. Liquid Chromatography 1 8 6 4 2 36.34 NL: 112.98 116.33 4.14 1.8E9 63.63 66.3 96.1 TIC MS 4.2 89.7 13.39 21491_.14 61.74 69.81 73.43 142.94 168.62 q_tc_col1 61.3 14.9 4.3 89.9 14.1 _gantasfi_v 46.29 6.71 81.87 16.36 173.72 _input 21.97 31.43 2.43 82.29 24.26 78.99 12.91 123.2 146.63 174.69 126. 131. 19.34 99.4 166.86 162.82 1. 161.31 14.44 134.76 12.17 1.12 137. 147.67 16.36 peptide separation C18 nanoflow HPLC 1 2 3 4 6 7 8 9 1 11 12 13 14 1 16 17 18 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)

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

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. 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 6 399.33 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 43.9283.377 479.331 92.34 12.383 43.9283 6.4398 772.292 7 4 479.331 92.34 286.218 64.4697 4 6 399.33 12.383 843.228.377 772.292 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. 1 187.113 261.16 3 371.2819 286.218 877.878 9.6296 146.96 2 64.4697 843.228 2 3 1 4 6 7 8 9 1 11 7. 1 187.113 261.16 371.2819 877.878 9.6296 146.96 818.9.377 818.9 6.4398 818.9 2 3 4 6 7 8 9 1 11 Tandem Mass Spectrometry (MS/MS) 3. Tandem Mass Spectrometry 1 Da 272.23;y2 peptide masses MS spectra cycles of MS and peptide fragment masses 1 MS spectrum = mass of the intact peptides 449 4 41 42 43 44 3 4 6 7 8 9 1 11 12 13 14 1 19.76;b1 1.139;y1 83.383;y 387.2;y3 486.297;y4 712.443;y6 1 2 3 4 6 7 8 9 1 11 12 13 14 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 #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 6.4398 21239_C1_sn8 479.331 #979 RT: 29.1 AV: 1 NL:.E4 T: ITMS + c NSI d Full ms2 2.33@cid. 92.34 [14.-111.] 12.383 1 9 399.33 8 7.377 6.4398 818.9 43.9283 479.331 92.34 21239_C1_sn8 #979 RT: 29.1 AV: 1 NL:.E4 12.383 T: ITMS + c NSI d Full ms2 2.33@cid. [14.-111.].377 1 43.9283 772.292 4 286.218 399.33 9 479.331 92.34 4 64.4697 843.228 6 12.383 818.9 772.292 8.377 7. 187.113 261.16 3 43.9283 371.2819 286.218 4 7 877.878 9.6296 2 64.4697 146.96 843.228 4 399.33 479.331 92.34 2 1 3 4 6 7 8 9 1 7. 11 6 12.383 1 187.113 261.16 772.292.377 3 371.2819 877.878 9.6296 43.9283 146.96 286.218 2 2 4 3 4 6 7 64.4697 8 9 843.228 399.33 1 11 1 4 7. 1 187.113 261.16 772.292 3 371.2819 877.878 9.6296 146.96 286.218 2 2 3 4 6 7 64.4697 8 9 843.228 1 11 1 7. 1 187.113 261.16 371.2819 877.878 9.6296 146.96 6.4398 818.9 6.4398 818.9 2 3 4 6 7 8 9 1 11 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

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

Overview Mass spectrometry-based proteomics: general workflow Identification of protein post-translational modifications (PTMs) Take-home messages are in red 17

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

I. Measurable mass difference: example from the lab Purified protein X mod Question: is the bacterial protein X modified by host enzymes? 837.61 2+ 21.6 8.9618 2+ incubation with host cell nuclear extract MS spectra Acetyl 42.1 42.112 Da Trimethyl 42.469 3.2 min +/- % DLALIKADLAEFEAR? 33.63 min Protein X gelband Ac + DLALIKADLAEFEAR? +/- % 836 838 84 842 844 846 848 2 4 6 8 86 862 864 Time (min)

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; 413.24 b; 26.32 b6; 4.42 b7; 7.46 I K A y8;.43 y9; 121.47 y1: 1149.4 y11; 1262.7 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; 121.47 y1: 1191. y11; 134.9 MS/MS spectra b4; 413.24 b; 26.32 b6; 696.43 b7; 7.46 I K-Ac A A K-Ac I 166.9 Ac DLALIKADLAEFEAR 8.41 842.36 1694.86 4 6 8 1 12 14 16

II. Pre-enrichment of modified peptides trypsin protein extract peptides LC-MS/MS

II. Pre-enrichment of modified peptides trypsin protein extract peptides pre-enrichment of modified peptides LC-MS/MS

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

Acknowledgements An Staes Evy Timmerman Delphi Van Haver Jarne Pauwels Teresa Maia Kris Gevaert Fabien Thery Lia Martina Katie Boucher