Supplementary Information. Top-down/bottom-up mass spectrometry workflow using dissolvable polyacrylamide gels

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1 Supplementary Information Top-down/bottom-up mass spectrometry workflow using dissolvable polyacrylamide gels Nobuaki Takemori,,* Ayako Takemori,, Piriya Wongkongkathep, Michael Nshanian, Rachel R. Ogorzalek Loo, ǁ Frederik Lermyte, and Joseph A. Loo,, ǁ,*. Proteo-Science Center, Division of Proteomics Research, Ehime University, Shitsukawa, Toon, Ehime, , Japan. Th e United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Ehime, , Japan. Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California 90095, USA ǁ. Department of Biological Chemistry, UCLA/DOE Institute for Genomics and Proteomics, and UCLA Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California 90095, USA. Department of Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk-Antwerp, Belgium * Correspondence should be addressed to N.T. (takemori@m.ehime-u.ac.jp) or J.A.L. (JLoo@chem.ucla.edu). Summary of Supporting Information Figure S-1. Tris-glycine SDS-PAGE using a BAC-crosslinked poly-acrylamide slab gel Figure S-2. MALDI-TOF MS analysis of standard proteins derived from BAC gels Figure S-3. Comparison of the performance of BAC-PAGE and gel electroelution for in-gel protein recovery Figure S-4. Evaluation of reproducibility of BSA recovery from BAC gel Figure S-5. LC-ESI-TOF MS analysis of HIST1H4A Figure S-6. Evaluation of cysteine alkylation by MS Table S-1. Composition of the gel solutions for BAC-PAGE S-1

2 Figure S-1. Tris-glycine SDS-PAGE using a BAC-crosslinked polyacrylamide slab gel The components of acrylamide solution for making tris-hcl gel are shown in Table S1. SDS-PAGE of protein extracts from the Drosophila brain tissue (10 μg total proteins) was carried out using tris/glycine/sds buffer (BioRad). The separated proteins were visualized with CBB. S-2

3 Figure S-2. MALDI-TOF MS analysis of standard proteins derived from BAC gels Standard proteins (BSA and HIST1H3A) were first separated by BAC-PAGE, and extracted by the protocol described in Fig.3a. Purified intact proteins were subjected to MALDI MS analysis using α-cyano-4-hydroxycinnamic acid as a matrix. MS spectra were acquired on a MALDI-TOF MS instrument (AXIMA TOF2: Shimadzu Corporation, Kyoto, Japan) in positive mode. *: observed protein ions. S-3

4 Figure S-3. Comparison of the performance of BAC-PAGE and gel electroelution for in-gel protein recovery (a) A crude protein extract from Drosophila brain (10 μg total protein) was first separated by SDS-PAGE using a Bis-crosslinked polyacrylamide gel. Gel-recovered proteins through electroelution were further subjected to 4-12% NuPAGE gel electrophoresis. *: Drosophila brain extracts (5 μg total protein/lane). Bis: Bis-crosslinked 8% (w/v) acrylamide gel. (b) The number of identified proteins with different protein recovery procedures. Gel-recovered proteins were digested with trypsin and subjected to LC-MS/MS analysis. S-4

5 Figure S-4. Evaluation of reproducibility of BSA recovery from BAC gel (a) Schematic diagram of the experiment performed to evaluate the reproducibility of in-gel protein recovery. (b) SDS-PAGE image of BSA using 4-12% NuPAGE. *: original BSA sample (2 μg/lane); Lane #1-5: recovered BSA. (c) Quantitative evaluation of BSA recovery from BAC gel using Qubit protein assay kit. S-5

6 Figure S-5. LC-ESI-TOF MS analysis of HIST1H4A. Comparison of LC-MS (Agilent TOF MS) of histone standard protein. S-6

7 a b Sequence Modifications Cleavages Prec m/z Theor m/z Theor z DAFLGSFLYEYSR FKDLGEEHFK HLVDEPQNLIK KQTALVELLK missed K-Q@ KVPQVSTPTLVEVSR missed K-V@ LCVLHEK Carbamidomethyl(C)@ LFTFHADICTLPDTEK Carbamidomethyl(C)@ LGEYGFQNALIVR Deamidated(N)@ LGEYGFQNALIVR LKPDPNTLCDEFKADEK Deamidated(N)@6; Carbamidomethyl(C)@9 missed K-A@ LVNELTEFAK QTALVELLK QTALVELLKHKPKATEEQLK missed K-H@9; missed K-A@ RPCFSALTPDETYVPK Carbamidomethyl(C)@ TVMENFVAFVDK Oxidation(M)@ VPQVSTPTLVEVSRSLGKVGTR missed R-S@14; missed K-V@ YICDNQDTISSK Carbamidomethyl(C)@ YLYEIAR e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 EYEATLEECCAK QNCDQFEK Q1: Q3: [y5] Q1: Q3: [y5] Q1: Q3: [y6] Q1: Q3: [y6] 5.2e5 4.8e5 4.4e5 4.0e5 3.6e5 3.2e5 2.8e5 2.4e5 2.0e5 1.6e5 1.2e5 8.0e4 4.0e e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 CCTESLVNR Q1: Q3: [y5] Q1: Q3: [y5] YICDNQDTISSK Q1: Q3: [y8] Q1: Q3: [y8] c 1.4e4 1.2e4 1.0e4 0.8e4 0.6e4 0.4e4 0.2e4 0.0 % Intensity TCVADESHAGCEK Q1: Q3: [y5] Q1: Q3: [y5] (M+2H) (M+H) e e m/z Figure S-6. Evaluation of cysteine alkylation by MS. (a) After alkylating the cysteines in BSA with iodoacetamide, we performed BAC-PAGE, digestion, and searched for acrylamide adducts by LC-MS/MS. Obtained MS/MS spectra were searched by the ProteinPilot (SCIEX) using the following parameters: cys alkylation, iodoacetamide with other cysteine modifications possible; digestion, trypsin; processing parameters, biological modification; and search effort, through ID. Table enumerates identified BSA peptides as reported by the ProteinPilot. In bottom-up analysis of the tryptic digest, no acrylamide adduct was observed. (b) We further evaluated the alkylated cysteine residues by selected reaction monitoring. Although partial acrylamide adduction was observed in BSA peptide YICDNQDTISSK, cysteine residues in 4 other peptides were completely alkyated by iodoacetamide. : iodoacetamide; : acrylamide. (c) In addition to iodoacetamide, 4-vinyl pyridine is also effective for alkylating cysteines. Hen egg white lysozyme (4 disulfide bonds, MW=14305) was reduced with dithiothreitol and alkylated with 4-vinyl pyridine. After acetone precipitation, this MALDI mass spectrum was obtained with 2,5-dihydroxybenzoic acid as matrix. The singly-charged ion at m/z corresponds to the reduced protein adding 8 vinyl pyridine molecules (predicted m/z). The shoulder at m/z is reduced protein with 7 vinyl pyridines. S-7

8 Table S-1. Composition of the gel solutions for BAC-PAGE a) Tris-acetate gel Stock solutions Acrylamide solution A: 30% (w/v) Acrylamide/0.5% (w/v) BAC Acrylamide solution B: 30% (w/v) Acrylamide/0.5% (w/v) BAC/0.2% (w/v) Bis Stacking Gel 4% Acrylamide 6% Acrylamide Resolving Gel 8% Acrylamide 10% Acrylamide 1300 μl 1733 μl 2167 μl 320 μl 1.0 M Tris-Acetate (ph 7.0) 480 μl 1300 μl 1300 μl 1300 μl 10% (w/v) SDS 24 μl 68 μl 68 μl 68 μl 1.5% (w/v) Ammonium persulfate 60 μl 214 μl 214 μl 214 μl 30%(v/v) Glycerol 800 μl TEMED 2.4 μl 65 μl 65 μl 65 μl Distilled water Bring to final volume (2.4 ml) with distilled water Bring to final volume (6.5 ml) with distilled water b) Tris-HCl gel Stock solutions Acrylamide solution A: 30% (w/v) Acrylamide/0.5% (w/v) BAC Stacking Gel Resolving Gel 4% Acrylamide 10% Acrylamide 320 μl 2167 μl 1.5 M Tris-HCl (ph 8.8) 1300 μl 0.5 M Tris-HCl (ph 6.8) 600 μl 10% (w/v) SDS 24 μl 68 μl 1.5% (w/v) Ammonium persulfate 60 μl 214 μl 30%(v/v) Glycerol 800 μl TEMED 2.4 μl 65 μl Distilled water Bring to final volume (2.4 ml) with distilled water Bring to final volume (6.5 ml) with distilled water S-8