Project PXD005183

PRIDE Assigned Tags:
Technical Dataset

Summary

Title

Systematic Evaluation of Protein Reduction and Alkylation reveals Massive Unspecific Side Effects by Iodine-containing Reagents

Description

In this study, we systematically evaluated reduction and alkylation of proteins using three reducing agents (DTT, TCEP, and BME) in combination with four alkylation agents (IAA, IAC, AA, and CAA). We tested these conditions using strong anion exchange (SAX) fractionated in-solution digests and in-gel digested fractions of samples enriched for cytosolic proteins of HeLa cells. We analyzed the data using Proteome Discoverer 2 in combination with the MASCOT search engine with different combinations of variable and fixed modifications as well as error tolerant and mass tolerant searches. Furthermore, we compared IAA and AA alkylated samples by dimethyl labeling and performed multi stage activation triggered by the neutral loss of alkylated methionine side chains. This led to the identification of prominent offside alkylation due to iodine containing reagents at tyrosine, serine, threonine, histidine, lysine, methionine, aspartic and glutamic acid as well as the peptide N-terminus with varying abundances based on the reduction and alkylation reagents used.

Sample Processing Protocol

In-solution reduction, alkylation, and digestion: For each experimental condition, 100 µg of protein were used. Protein reduction was performed for 30 minutes at 56°C with 5 mM DTT, 5 mM TCEP, or 10 mM BME, respectively. Subsequent alkylation was performed in the dark for 30 minutes at 23°C with 20 mM IAA, IAC, AA, or CAA, respectively. Samples were quenched by addition of DTT, TCEP, or BME, and digested in-solution by filter aided sample preparation (FASP), using 30 kD molecular weight cutoff filters (Microcons, Millipore) as described elsewhere (18). Briefly, samples were mixed with 200 µl of 8 M urea in Tris/HCl, pH 8.5, loaded in the filtration devices and centrifuged at 14,000 x g for 15 minutes. 4 additional centrifugation steps were performed using buffer containing 8 M urea followed by 3 steps with 100 µl 0.05 M NH4HCO3. Finally, 1 µg trypsin was added to each sample in 60 µl 0.05 M NH4HCO3 and incubated in a wet chamber overnight at 37°C (enzyme/protein ratio 1:100). Peptides were eluted from the filtration devices and desalted using Oasis HLB 1 cc 10 mg cartridges. The eluate was dried using a vacuum centrifuge, re-suspended in 50 µl of water, and dried again. Subsequently SAX fractionation was performed as described elsewhere (8). Briefly, anion exchange microcolumns were manufactured by stacking 12 layers of 3M Empore anion exchange disks into a 200 µl micropipette tip. Microcolumns were equilibrated by consecutive centrifugation steps at 5000 x g for 3 minutes using 100 µl MeOH, 100 µl 1 M NaOH, and 100 µl Britton & Robinson buffer (BR buffer, 20 mM acetic acid, 20 mM phosphoric acid, and 20 mM boric acid) adjusted to pH 11. Peptides were dissolved in 100 µl BR buffer, at pH 11, loaded on the microcolumns and eluted stepwise with 100 µl of BR buffer adjusted to pH 8, 6, 5, 4, and 3. Fractions were collected on Stage tips (19) containing 6 layers of 3M Empore C18 membrane. Stage tips were washed with 50 µl 0.1% trifluoroacetic acid (TFA) and peptides eluted with 40 µl 60% acetonitrile (ACN), dried in a vacuum centrifuge, and re-suspended in 20 µl 5% ACN, 5% FA. In-gel reduction, alkylation, and digestion: For each condition 50 µg of protein, mixed in a 1:4 (v/v) ratio with sample loading buffer (0.25 M Tris/HCl pH 6.8, 8% SDS, 40% Glycerol, and 0.004% Bromophenol blue), was loaded on a 10% SDS-PAGE gel. Electrophoresis was performed, the gels washed 2 times with ddH2O, and stained using Coomassie brilliant blue (Page Blue Protein Staining Solution, Thermo Scientific) overnight. Gels were destained using ddH2O, the same gel section excised for each sample, cut into cubes of ~1 mm3, and transferred to 1.5 ml micro tubes. Gel pieces were destained twice using 1 ml of 30% acetonitrile (ACN)/0.07 M NH4HCO3 at 25°C, 800 rpm for 30 minutes and reduced using 100 µl of either 20 mM DTT, 20 mM TCEP, or 40 mM BME in 0.1 M NH4HCO3 at 56°C for 45 minutes. After removal of the reducing reagent, 100 µl of 55 mM IAA, IAC, AA, or CAA in 0.1 M NH4HCO3 was added and incubated at 23°C for 30 minutes in the dark followed by washing with 0.1 M NH4HCO3 at 800rpm, 23°C for 15 minutes. Subsequently, gel pieces were dehydrated by ACN and dried using a vacuum centrifuge. To each sample, 1µg of trypsin (Promega) in 0.1 M NH4HCO3 was added and incubated overnight at 37°C. Peptides were extracted from the gel pieces using an optimized protocol (6). Briefly, gel pieces were incubated consecutively with 50 µl 0.1% TFA, 50% ACN, 50 µl 0.1 M NH4HCO3, and 100 µl 100% ACN at 25°C, 800 rpm for 15 minutes. Supernatants were combined, dried using a vacuum centrifuge, re-suspended in 100 µl 0.01% acetic acid, 3% ACN, and desalted using C18 stage tips (19). Peptides were eluted twice using 20 µl 0.5% acetic acid 80% ACN, eluate fractions dried using a vacuum centrifuge and re-suspended in 20 µl 5% ACN, 5% formic acid (FA). Dimethyl labeling: The same regions of the SDS-PAGE gel were cut as for the other analyses, proteins reduced, alkylated, and digested in-gel using trypsin and triethylammonium bicarbonate (TEAB) buffer. Extracted peptides were dried using a vacuum centrifuge, re-suspended in 0.1 M TEAB, and either labeled with light-, intermediate- or heavy-formaldehyde as described elsewhere (20). Briefly, dried samples were re-suspended in 100 µl 0.1 M TEAB, 4 µl of 4% formaldehyde (CH2O, CD2O or 13CD2O) and 4 µl 0.6 M cyanoborohydride (NaBH3CN or NaBD3CN) added and the labeling reaction was allowed to proceed for 1h at 23°C. After addition of 16 µl 1% ammonia and 8 µl, FA samples were mixed, desalted using Oasis cartridges, and dried using a vacuum centrifuge.

Data Processing Protocol

Data analysis: Thermo *.raw files were searched against Uniprot human (release 2016_01) using Mascot 2.5.1 with Proteome Discoverer 2.1.0.81. Depending on the searches different combinations of modifications were used: 1. Carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at cysteine and oxidation at methionine as variable modifications (Supplementary Table 1 & 2) 2. Carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at all amino acids and oxidation at methionine as variable modifications (Supplementary Table 3) 3. Carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at cysteine as fixed modification and at tyrosine, serine, threonine, histidine, lysine, aspartic and glutamic acid as well as the peptide N-terminus as variable modification as well as oxidation at methionine as variable modifications (Supplementary Table 4 & 5) 4. Dimers of Carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at all amino acids and oxidation at methionine as variable modifications; Carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at cysteine as fixed modification and at tyrosine, serine, threonine, histidine, lysine, aspartic and glutamic acid as well as the peptide N-terminus as variable modification as well as oxidation at methionine as variable modifications (Supplementary Table 6 & 7) 5. Dimethylation (light, medium, heavy) as variable modification at lysine and peptide N-termini, carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at cysteine and oxidation at methionine as variable modifications (Supplementary Table 8) 6. Dethiometyl at methionine, carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at cysteine and oxidation at methionine as variable modifications (Supplementary Table 10 & 11) 7. Dethiometyl at methionine, carbamidomethyl (IAA, CAA), carboxymethyl (IAC) or propionamide (AA) at cysteine and methionine and oxidation at methionine as variable modifications (Supplementary Table 12) For all searches, the precursor mass tolerance was set to 10 ppm and the fragment ion tolerance to 0.8 Da. Trypsin was selected as enzyme and 2 missed cleavage sites were allowed. Peptides were exported with a false discovery rate of 1% at the peptide level and further processed using MS Excel. For error tolerant searches, Mascot result files were opened in a browser window in the peptide summary format, the top 20 protein hits selected for error tolerant searching. For mass tolerant searches, *.raw files were converted to *.mgf files using Proteome Discoverer. The header of the *.mgf files was modified as follows: peptide mass tolerance: 250 Da; fragment ion tolerance: 0.8 Da; search type: SQ; fixed modifications: carbamidomethyl, carboxymethyl, or propionamide, respectively, at cysteine residues depending on the alkylation reagent. Searches were performed with and without oxidized methionine as variable modification. MSMS ion searches using the modified *.mgf files were performed using Mascot with the following parameters: Uniprot human, trypsin with 2 missed cleavage sites, and ESI-TRAP as instrument. For error and mass tolerant searches, data were exported to MS Excel using the export function of Mascot in the browser window applying a peptide score cutoff of 30 (Supplementary Table 9)

Contact

Torsten Mueller, DKFZ
Dominic, Winter, University of Bonn, Institute of Biochemistry and Molecular Biology, Germany ( lab head )

Submission Date

10/04/2017

Publication Date

30/05/2017

Tissue

Not available

Instrument

LTQ Orbitrap Velos

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Müller T, Winter D. Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents. Mol Cell Proteomics. 2017 May 24. pii: mcp.M116.064048 PubMed: 28539326