Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for

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1 Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies Zhen Wu, Jichang Huang, Jingnan Huang, Qingqing Li, Xumin Zhang *, State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 8, China Supplemental Information Lys-C/Arg-C Digestion Protocol S-, S- Figure S-. Analysis of wrong cleavage sites by Lys-C, Arg-C and trypsin when searched as trypsin. S- Figure S-. The Venn diagram of peptides identified from the four different digestion approaches. S- Figure S-. The reproducibility of identified proteins from the four approaches in triplicate experiments. S-7 Figure S-. Analysis of wrong cleavage sites by different digestion approaches. S-8 Figure S-. The reproducibility of detected peptides from four approaches by DIA acquisition methods in triplicate experiments. S-9 Figure S-. Principle component analysis (PCA) of the quantification results by different digestion approaches. S- Figure S-7. The correlation of the intensity of peptides from two replicates of each digestion method. S- Table S-. The identified peptides of Lys-C, Arg-C and trypsin when searched as trypsin with missed cleavage sites. S-

2 Table S-. Identification results by different digestion approaches in triplicate experiments. Table S-. DIA quantification results at peptide level by different digestion approaches. S-

3 Lys-C/Arg-C Digestion Protocol Sample preparation TIMING h ) Cell lysate. Add volumes of lysis buffer ( M guanidine hydrochloride, mm TEAB) to the collected cells after washing three times with PBS buffer, and sonicate for min ( s sonication with s intervals). ) Collection. Collect supernatant via centrifugation at, g for min at C. ) Protein determination. Determine the protein concentration using Bradford assay. ) Reduction. Add mm DTT and then incubate at 7 C for min. ) Alkylation. Add mm acrylamide and incubate for h at room temperature. ) Quenching. Add DTT to a final concentration of mm to quench excess acrylamide. 7) Dilute the proteins to mg/ml with lysis buffer. Digestion TIMING - h 8) Buffer displacement. Transfer samples from Eppendorf tubes to Microcon YM- filters and centrifuged at,8 g for three-time buffer displacement, the first and second time with mm Tris-HCl (ph 8.) and % ACN, and the last time with mm Tris-HCl (ph 8.). This step takes to h, depending on sample volume and protein concentration. 9) First-step Lys-C digestion. Add digestion buffer ( mm Tris-HCl, ph 8.) to reach the protein concentration of mg/ml. Add Lys-C at a ratio of enzyme/protein as : and incubate at 7 C for h. ) Second-step Arg-C digestion. Add DTT, EDTA and calcium chloride to reach the recommended concentration in the supplier s protocol ( mm DTT,. mm EDTA and 8. mm calcium chloride). Add Arg-C at a ratio of enzyme/protein as : and incubate at 7 C for h. S-

4 Sample collection TIMING - h ) After digestion, collect the filtrate via centrifugation at,8 g. To minimize sample loss, wash the filter twice with % ACN and collect the filtrates via centrifugation at,8 g. Pool the three filtrates together. ) Remove the remaining ACN and concentrate sample to about mg/ml by a Speedvac. S-

5 Occurrence (%) Occurrence (%) Occurrence (%) A Lys-C(trypsin) B Arg-C(trypsin) Database Lys-C(trypsin) Database Arg-C(trypsin) C Trypsin Database Trypsin Figure S-. Analysis of wrong cleavage sites by Lys-C, Arg-C and trypsin when searched as trypsin. The occurrences of wrong cleavage sites and amino acids in human protein database (the left y-axis) and the relative occurrences (the right y-axis) for (A) Lys-C, (B) Arg-C and (C) trypsin. S-

6 Lys-C/trypsin Trypsin (:) Lys-C/Arg-C 8 Trypsin (:) Figure S-. The Venn diagram of peptides identified from the four different digestion approaches. S-

7 A Lys-C/Arg-C digestion 9 (7.%) 7 B Lys-C/trypsin digestion (9.%) 7 8 C Trypsin (:) digestion D Trypsin (:) digestion (7.%) (.9%) 8 Figure S-. The reproducibility of identified proteins from the four approaches in triplicate experiments. (A) Lys-C/Arg-C, (B) Lys-C/trypsin, (C) trypsin (:) and (D) trypsin (:). S-7

8 Occurrence (%) Occurrence (%) Occurrence (%) Occurrence (%) A Lys-C/Arg-C B Lys-C/trypsin Database Lys-C/Arg-C Database Lys-C/trypsin C Trypsin(:) D Trypsin(:) Database Trypsin(:) Database Trypsin(:) Figure S-. Analysis of wrong cleavage sites by different digestion approaches. The occurrences of wrong cleavage sites and amino acids in human protein database (the left y-axis) and the relative occurrences (the right y-axis) for (A) Lys-C/Arg-C, (B) Lys-C/trypsin, (C) trypsin (:) and (D) trypsin (:). S-8

9 A Lys-C/Arg-C digestion (7.%) 8 B 79 Lys-C/trypsin digestion 9 8 (7.7%) C Trypsin (:) digestion 7 89 (7.%) D Trypsin (:) digestion 89 7 (7.%) Figure S-. The reproducibility of detected peptides from four approaches by DIA acquisition methods in triplicate experiments. (A) Lys-C/Arg-C, (B) Lys-C/trypsin, (C) trypsin (:) and (D) trypsin (:). S-9

10 Figure S-. Principle component analysis (PCA) of the quantification results by different digestion approaches. S-

11 B Lys-C/trypsin replicate Lys-C/trypsin R²=.99 C Trypsin (:) replicate Trypsin (:) D Trypsin (:) replicate Trypsin (:) Trypsin (:) replicate R²=.9 Lys-C/Arg-C replicate Trypsin (:) replicate Lys-C/Arg-C replicate R²=.889 Lys-C/trypsin replicate R²=.99 Trypsin (:) replicate R²=.99 Lys-C/trypsin replicate R²=.9 R²=.9 Lys-C/Arg-C replicate Trypsin (:) replicate R²=.9 Lys-C/trypsin replicate R²=.9 Lys-C/Arg-C replicate Lys-C/trypsin replicate Lys-C/trypsin replicate Trypsin (:) replicate R²=.9 Trypsin (:) replicate Lys-C/Arg-C replicate R²=.98 Trypsin (:) replicate Lys-C/Arg-C replicate Lys-C/Arg-C Trypsin (:) replicate A Trypsin (:) replicate R²=.89 Trypsin (:) replicate Figure S-7. The correlation of the intensity of peptides from two replicates of each digestion method. (A) Lys-C/Arg-C, (B) Lys-C/trypsin, (C) trypsin (:) and (D) trypsin (:). S-