An Antibody-free Approach for the Global Analysis of. Protein Methylation

Transcription

1 Supplemental materials to An Antibody-free Approach for the Global Analysis of Protein Methylation Keyun Wang,, Mingming Dong,, Jiawei Mao,, Yan Wang,, Yan Jin,, Mingliang Ye,, *,,, Hanfa Zou CAS Key Lab of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences Dalian , China University of Chinese Academy of Sciences, Beijing , China * To whom correspondence should be addressed: (M.L. Ye) Phone: Fax: mingliang@dicp.ac.cn. Table of Contents Supplementary Experimental Procedures Supplementary Figures Supplementary Tables 1

2 Supplementary Experimental Procedures Materials and Reagents. L-Lysine, L-Arginine, L-Methionine-(methyl- 13 C,D 3 ), formic acid, trifluoroacetic acid (TFA), 2, 5-dihydroxybenzoic acid (2, 5-DHB), dithiothreitol (DTT), iodoacetamide (IAA), bovine serum albumin (BSA), Protease inhibitor cocktail, Triton X-100, Sodium chloride, Ammonium bicarbonate (NH 4 HCO 3 ) and acetic acid were obtained from Sigma-Aldrich; EIAQDF-K-TDLR, EIAQDF-K(Me1)-TDLR, EIAQDF-K(Me2)-TDLR, EIAQDF-K(Me3)-TDLR, SG-R(Me1)-GGNFGFGDSR and N-R(Me2S)-GAGGFGGGGGTR were custom-synthesized from Scilight Biotechnology. Methanol and acetonitrile were chromatographic grade from Merck (Darmstadt, Germany); Tris, EDTA, and urea are guaranteed reagent, all other chemicals, including phosphorous acid, boric acid and calcium chloride were of analytical grade. Lysyl endopeptidase (Lys-C), trypsin (TPCK-modified, MS grade) and clostripain (Arg-C) were purchased from Wako Chemicals, Hualishi Technologies and Worthington Biochemical respectively. Deionized water used for all experiments was purified with a Milli-Q water system (Millipore). Degreasing cotton was purchased from Shanghai Shengbeinuo Medical Apparatus Co., Ltd. Digestion of synthetic methylated peptides. 50 µg synthetic methylated peptide (EIAQDF-K-TDLR, EIAQDF-K(Me1)-TDLR, EIAQDF-K(Me2)-TDLR, EIAQDF-K(Me3)-TDLR, SG-R(Me1)-GGNFGFGDSR, N-R(Me2S)-GAGGFGGGGGTR) were dissolved in 50 µl 50mM NH 4 HCO 3 (ph 8.1). After the addition of trypsin, the resulting solution was incubated for 16 h for digestion at 37 C. Quench the digestion by acidification with TFA to 1% (vol/vol) on ice and the sample was then subjected to MALDI-TOF MS analysis. 50 µg synthetic methylated peptide (EIAQDF-K-TDLR, EIAQDF-K(Me1)-TDLR, EIAQDF-K(Me2)-TDLR, EIAQDF-K(Me3)-TDLR, SG-R(Me1)-GGNFGFGDSR, N-R(Me2S)-GAGGFGGGGGTR) were dissolved in 50 µl 50mM NH 4 HCO 3 (ph 8.1). After the addition of Trypsin (1:50) and Lys-C (1:50) at an enzyme-to-protein ratio of 1:50, the resulting solution was incubated for 16 h for digestion at 37 C. Then 500 2

3 mm CaCl 2 was added to above solution to a final concentration of 5mM followed with the addition of 10 activation buffer (50mM Tris-HCl (ph 7.8), 50mM DTT and 2mM EDTA) to a final concentration of 1. Then Arg-C was added at an enzyme-to-protein ratio of 1:50 and incubate for 16 h for further digestion at 37 C. The digestion was quenched by acidification with TFA to 1% (vol/vol) on ice and the sample was subjected to MALDI-TOF MS analysis. Optimization of digestion conditions for complex sample. The FASP sample preparation protocol(1) with modifications was applied to optimize the conditions to digest the complex protein sample. Before digestion,100 µg proteins from hm-silac HepG2 cells were reduced by 20 mm DTT at 37 for 2 h and alkylated by 40 mm iodoacetamide in the dark at room temperature for 40 min. The solution was transferred to a 10-kDa-MWCO filter and centrifuge it at 14,000g for 15 min to remove the urea lysis buffer followed with washing three time with 50mM NH 4 HCO 3 (ph 8.1). After adding 100 µl 50mM NH 4 HCO 3 (ph 8.1) to the 10-kDa-MWCO filter, trypsin was added at an enzyme-to-protein ratio of 1:50 and incubate it for 16 h for digestion at 37 C. This method was termed one step one enzyme (Trypsin). The second method was termed as one step two enzymes (Trypsin & Lys-C). The procedure was the same except two enzymes, Trypsin and Lys-C, were added together. The third method was termed as two step multiple enzymes (Trypsin & Lys-C & Arg-C). In this method, the sample generated by Trypsin & Lys-C (the second method) was further processed as following. The sample was centrifugated at speed of 14,000g to remove the Trypsin and Lys-C. Collect the filtrate and add 500 mm CaCl2 to above filtrate to a final concentration of 5mM, then add 10 activation buffer (50mM Tris-HCl (ph 7.8), 50mM DTT and 2mM EDTA) to a final concentration of 1. Then add Arg-C at an enzyme-to-protein ratio of 1:50 and incubate for 16 h for further digestion at 37 C. Collect sample 3 (Trypsin & Lys-C & Arg-C). The fourth method was two step one enzyme. In this method, trypsin was added at an enzyme-to-protein ratio of 1:25 and incubate it for 16 h for digestion at 37 C. The sample was centrifugated at speed of 14,000g to remove the Trypsin. Collect the filtrate and add trypsin at an enzyme-to-protein ratio of 1:50, then incubate for 16 h for further 3

4 digestion at 37 C. Quench all the above digestions by acidification with TFA to 1% (vol/vol) on ice and desalt the resulting peptides with 10mg HLB cartridge. 4

5 Figure. S-1. The MALDI mass spectrometric analyses of methylated (R) peptides after the digestion with trypsin. (A) Mono-methylated (R) peptide, SG-R(Me1)-GGNFGFGDSR; (B) Di-methylated (R) peptide, N-R(Me2S)-GAGGFGGGGGTR. 5

6 Figure. S -2. The MALDI mass spectrometric analyses of methylated peptides after the digestion with multiple enzymes (Trypsin, Lys-C and Arg-C). (A) Mono-methylated (K) peptide, EIAQDF-K(Me1)-TDLR; (B) Di-methylated (K) peptide, EIAQDF-K(Me2)-TDLR; (C) Tri-methylated (K) peptide, EIAQDF-K(Me3)-TDLR; (D) Mono-methylated (R) peptide, SG-R(Me1)-GGNFGFGDSR; (E) Di-methylated (R) peptide, N-R(Me2S)-GAGGFGGGGGTR. 6

7 Figure. S-3. Photographs of the SCXtip and the constructing process. 7

8 Figure. S-4. The MALDI mass spectrometric analysis of a methylated peptide mixture eluted with different washing buffers from the SCXtip. Sample: Mixture of five synthesized methylated peptides (1: EIAQDF-K(Me1)-TDLR; 2: EIAQDF-K(Me2)-TDLR; 3: EIAQDF-K(Me3)-TDLR; 4:SG-R(Me1)-GGNFGFGDSR; 5: N-R(Me2S)-GAGGFGGGGGTR). (A) Direct analysis of the mixture; (B) The flow-through during the sample loading process; (C) Washing with 90% ACN, 5mM BRUB buffer (ph 9); (D) Washing with 85% ACN, 5mM BRUB buffer (ph 9); (E) Washing with 80% ACN, 5mM BRUB buffer (ph 9); (F) Washing with 65% ACN, 5mM BRUB buffer (ph 9); (G) Washing with 30% ACN, 5mM BRUB buffer (ph 12). 8

9 Figure. S-5. The MALDI mass spectrometric analysis of a mixture of 0.05mg methylated peptides (EIAQDF-K(Me1)-TDLR, EIAQDF-K(Me2)-TDLR, EIAQDF-K(Me3)-TDLR, SG-R(Me1)-GGNFGFGDSR, N-R(Me2S)-GAGGFGGGGGTR) and 1mg BSA tryptic digest during the loading to the SCXtip. (A) Direct analysis of the mixture; (B) The flow-through of the sample loading process. * denotes the methylated peptides. 9

10 Figure. S-6. The identification results of the methylproteomic analysis. (A) Percentage of unique methylated peptides identified in flow-through fraction, washing fraction, different elution fractions and untreated sample (labeled percentage denotes the averaged result of 3 replicates). (B) The overlap of identified methylation sites between three independent methylproteomic analysis. (C) The overlap of methylated peptide between different elution fractions. 10

11 Figure. S-7. Sequence logo representation of the Methyl-R sites in the high confidence group, low confidence group and in the control dataset. Figure. S-8. Motifs enriched around the identified methylation (R) residues. 11

12 Figure. S-9. The percentage of acidic residues in eluted fractions, flow-through and washing fractions. 12

13 Table S-1. Percentage of miscleavage after digestion by single or multiple enzymes. (Protein samples were digested with Trypsin (1:50), Trypsin(1:50)&Lys-C(1:50), Trypsin(1:50)&Lys-C(1:50)&Arg-C(1:50) or Trypsin(1:25)&Trypsin(1:50) to evaluate the mis-cleavage of each digestion approach.) Methods* Endopeptidase Replicate Replicate Replicate Averaged One step one enzyme Trypsin (1:50) 13.65% 14.29% 14.96% 14.3% One step two enzymes Trypsin(1:50) & Lys-C(1:50) 8.11% 7.55% 7.31% 7.66% Two step Trypsin(1:50) & multiple Lys-C(1:50) 4.84% 4.91% 4.79% 4.85% enzymes & Arg-C(1:50) Two step one enzyme Trypsin(1:25) & Trypsin(1:50) 13.76% 13.89% 13.43% 13.69% * Refer to Optimization of digestion conditions for complex sample section in this supplementary material. Table S-2. Percentage of histidine containing peptides identified in flow-through, washing and elution fractions, and the untreated sample, the fractions with peptides with over +3 charges in conventional SCX approach. Replicate 1 Replicate 2 Replicate 3 Averaged Percentage of histidine containing peptides in flow-through fraction 4.3% (282/6532) 4.4% (295/6643) 4.1% (274/6699) 4.3% Percentage of histidine containing peptides in washing fraction 33.0% (4167/12642) 32.9% (4228/12824) 33.7% (4304/12755) 33% Percentage of histidine containing peptides in elution fractions 24.5% (2923/11935) 24.3% (2879/11879) 24.2% (2955/12201) 24.3% Percentage of histidine containing peptides in untreated sample 18.3% (1679/9181) 18.2% (1686/9190) 18.7% (1741/9292) 18.5% Percentage of histidine containing peptides in the fractions with peptides with over +3 charges in conventional SCX approach(2) 60-70% 13

14 Table S-3. Modifications with mass difference identical to that of protein methylation without stable isotope labeling (by checking the Unimod Database, Accession Interim name Description Monoiostopic mass Composition 34 Methyl Methylation H(2) C 558 Asp Glu Asp Glu substitution H(2) C 571 Gly Ala Gly Ala substitution H(2) C 650 Ser Thr Ser Thr substitution H(2) C 671 Val Xle Val Leu/Ile substitution H(2) C 1162 Asn Gln Asn Gln substitution H(2) C 36 Di-Methylation Di-Methylation H(4) C(2) 255 Acetald+28 Acetaldehyde H(4) C(2) 280 Ethyl Ethylation H(4) C(2) 546 Ala Val Ala Val substitution H(4) C(2) 1061 Cys Met Cys Met substitution H(4) C(2) 37 Tri-Methylation Tri-Methylation H(6) C(3) 575 Gly Val Gly Val substitution H(6) C(3) 1047 Ala Xle Ala Leu/Ile substitution H(6) C(3) 1305 Propyl Propyl H(6) C(3) Table S-4. Modifications with mass difference close to that of heavy labeled protein methylation (by checking the Unimod Database, Accession Interim name Description Monoiostopic mass Composition 1079 Glu Phe Glu Phe substitution H(2) C(4) O(-2) 329 Methyl-Heavy Monomethylation H(-1) 2H(3) 13C 906 Lys MetOx Lys Met substitution and H(-3) C(-1) N(-1) O S sulfoxidation 987 Label:13C(6)15N(2)+Di Dimethyl 13C(6)15N(2) H(4) C(-4) 13C(6) methyl Silac label N(-2) 15N(2) 330 Dimethyl-Heavy Dimethylation H(-2) 2H(6) 13C(2) 958 Propargylamine propargylamine H(3) C(3) N O(-1) 859 MG-H1 Methylglyoxal-derived H(2) C(3) O hydroimidazolone 1414 Trimethyl:13C(3)2H(9) 3-fold methylation with fully H(-3) 2H(9) 13C(3) labelled methyl groups 1153 Met Trp Met Trp substitution H C(6) N S(-1) 14

15 Table S-5. Methylation settings for database searching. Modification types Residues Mono mass Positions Mono-H-methylation K, R anywhere Di-H-methylation K, R notcterm Tri-H-methylation K notcterm Heavy-methionine M anywhere Table S-6. Substitution rate of methionine for evaluating hm-silac labeling efficiency. Replicate 1 Replicate 2 Replicate 3 Averaged Substitution rate of methionine 98.06% 98.71% 98.61% 98.46% Table S-7. Number of unique methylated peptides and total peptides identified in different elution fractions (different replicates). Elution 1 Elution 2 Elution 3 Elution 4 Elution 5 Replicate 1 323/ / /885 96/892 46/834 Replicate 2 299/ / / /902 43/918 Replicate 3 317/ / / /842 50/1013 Table S-8. Identification results of global methylation analysis (methylation site). Methylation types Replicate_1 Replicate_2 Replicate_3 Sum Mono-methyl_KR Di-methyl_KR Tri-methyl_K All methylation types

16 Table S-9. LC gradient used in in-depth analysis of protein methylation. Time (min) Buffer A (%) Buffer B (%) Notes Sample loading Peptide separation Washing System equilibration References: 1. Wisniewski, J. R., and Rakus, D. (2014) Multi-enzyme digestion FASP and the 'Total Protein Approach'-based absolute quantification of the Escherichia coli proteome. J Proteomics 109, Uhlmann, T., Geoghegan, V. L., Thomas, B., Ridlova, G., Trudgian, D. C., and Acuto, O. (2012) A method for large-scale identification of protein arginine methylation. Mol Cell Proteomics 11,