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Technical Note
The impact of commonly used alkylating agents on artefactual peptide modification Peter G Hains, and Phillip J Robinson J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00022 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017
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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Proteome Research
The impact of commonly used alkylating agents on artefactual peptide modification Peter G. Hains1* and Phillip J. Robinson1
1- ProCan and the Cell Signalling Units, Children’s Medical Research Institute, The University of Sydney, 214 Hawkesbury Rd, Westmead, NSW Australia, 2145. ORCID PGH: 0000-0002-7276-1760 PJR: 0000-0002-7878-0313 KEYWORDS: Alkylation, cysteine alkylation, N-terminal alkylation, artefact, posttranslational modification
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ABSTRACT Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has been recently suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr and Tyr. Here, we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these was methionine oxidation, which increases to a maximum of 40% of all Met containing peptides compared to 2-5% with iodoacetamide. Increases were also observed for mono and dioxidised tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-choloraetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggest it is not the ideal alkylating reagent for proteomics.
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Journal of Proteome Research
Introduction Chemically-induced reduction and alkylation of disulphide bonds is an early and essential component of any proteomics sample preparation protocol. It aids in the denaturation and subsequent digestion of these proteins1. A wide diversity of chemicals is available to reduce and alkylate proteins. For reduction there is: dithiothreitol, β-mercoptoethanol, tris(2carboxyethyl)phosphine, 1,4-dithioerythritol and dithiobutylamine to name a few. Among the alkylating agents there is: iodoacetamide, iodoacetic acid, 4-vinylpyridine, 2chloroacetamide and N-ethylmaleimide amongst many others. Some of these reagents require reduction first, followed by a separate alkylation reaction, whereas others allow for simultaneous reduction and alkylation. Some protocols quench excess alkylating reagent by the subsequent addition of reducing agent. Among this variety, the most commonly employed reducing and alkylation agents and procedure is likely a two-step procedure, whereby dithiothreitol (DTT) is used to reduce the disulphide bonds, followed by iodoacetamide (IOA) to alkylate the free cysteines (Cys)1. The aim of these reactions is to alkylate only the Cys residues in peptides or proteins. However, side reactions can occur and it is possible to artefactually alkylate the N-terminus of proteins along with the following amino acids: Asp, Glu, His, Lys, Met, Ser, Thr and Tyr2– 5 . These unnatural modifications can lead to issues when computationally processing complex proteomics datasets3,4,6–9. Therefore, the use of other alkylating agents has been suggested. 2-Chloroacetamide (CA), in particular, shows a high degree of specificity towards Cys residues10. The mass addition using CA is identical to IOA, and the reaction conditions are unchanged, so there is little required to change existing protocols from IOA to CA. While IOA and CA were initially compared for alkylation of cysteines and the diglycinelike artefact10, a comprehensive comparison of other side-reactions was not reported. This paper evaluates the advantages and disadvantages of both IOA and CA when used for alkylation of proteins prior to enzymatic digestion and subsequent LC-MS/MS analysis. Experimental Procedures Sample preparation The isolation of organs from animals was done with approval from the Animal Care and Ethics Committee for the Children's Medical Research Institute, Sydney, Australia (project number C116). Rat testis were collected, placed in liquid nitrogen and transferred to -80ºC until required. Samples were processed in 2 groups of 8 over 3 separate experiments, for a total of 24 samples per condition. Tissue punches (1-2 mg) were taken whilst testis were on dry ice, placed into Barocycler tubes and processed by pressure cycling technology (PCT) using a modified version of the PCT-SWATH protocol11. 6 M urea, in 50 mM NH4HCO3, pH 7.8 was added to the tissue punches and a micropestle used to cap the tube12. The tissue was homogenised and cells lysed using 60 cycles of 45 kpsi for 50 sec, followed by 10 sec at atmospheric pressure at 35ºC in a Barocycler 2320EXT (Pressure Biosciences Inc, Boston, MA). Reduction and alkylation was performed during this process with either tris(2carboxyethyl)phosphine (TCEP) and IOA (Sigma ≥99% purity), or TCEP and CA (Sigma ≥98% purity, Lot: SLBK0729V)13,14. The TCEP was used at a final concentration of 10 mM and the IOA or CA were used at a final concentration of 40 mM. Following homogenisation, proteins were digested using Lys-C (Wako) for 45 cycles of 20 kpsi for 50 sec, followed by 10 sec at atmospheric pressure at 35ºC in a Barocycler 2320EXT. The micropestle was replaced with a microcap (150 µL), the urea diluted to 1.1 M with 50 mM NH4HCO3, pH 7.8 and the peptides further digested with trypsin (Promega) for 90 cycles of 20 kpsi for 50 sec,
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followed by 10 sec at atmospheric pressure at 35ºC in a Barocycler 2320EXT. In a one-off experiment to compare proteolytic digestion with Barocycler technology and digestion at atmospheric pressure, 16 samples (rat testis punches of 1-2 mg) were lysed in the Barocycler as outlined above. Eight of these samples (4 alkylated with IOA and 4 with CA) were digested with Lys-C and trypsin as indicated above (45 then 90 min respectively). The remaining 8 samples (4 alkylated with IOA and 4 with CA) were treated as above for the Lys-C and trypsin digests except that the Lys-C digest as at 35ºC, for 4 hrs at 1 atm and the trypsin digest was at 35ºC for 16 hrs at 1 atm. In a separate experiment, CA was sourced from three different suppliers and samples were treated as outlined above using the PCT-SWATH protocol. A total of 5 samples (rat testis, 1-2 mg punches) were processed per CA supplier in a one off experiment. The Sigma CA was as above, Merck CA, ≥97% purity, Lot: S7204112707 and Wako CA, >95% purity, Lot: LKM0253. All digests were made up to a final volume of 1 mL with 0.5 % (v/v) formic acid in water and passed through an Empore™ C18 4 mm/1 mL SPE cartridge to concentrate and desalt the peptides. Peptides were eluted and dried in a speed-vac, resuspended in 20 µL 0.5% (v/v) formic acid in water and their concentration assessed using A280 with an Implen NP80 nanophotometer. Two micrograms of lysate were injected per sample onto the LC-MS/MS system. Mass Spectrometry and data analysis Samples were analysed by LC-MS/MS using a Sciex 6600 mass spectrometer coupled to an Eksigent Ekspert nanoLC 415 HPLC system. Samples were loaded onto a trapping column (SGE C18G, 200 Å, 3 µm, 10 mm × 300 µm) before separation on an analytical column (SGE C18G, 200 Å, 3 µm, 250 mm × 300 µm) at a flow rate of 5 µL/min for a total run time of 90 min. The top 30 peptides were subjected to MS/MS analysis and searched against a rat only sequence database, extracted from Uniprot (37,596 entries), using ProteinPilot 5.015 with settings of: urea denaturation, Thorough ID search and FDR analysis (set at 1% global FDR for protein identification). The ProteinPilot FULL report option was used since it includes considerable information not included in the standard reporting template. Cys-CAM was used for Cys modification when either IOA or CA were used for alkylation. The Thorough ID search setting automatically searches for many known PTMs, including off-target alkylation of the N-terminus and Asp, Glu, His, Lys, Ser, Thr and Tyr residues. Additionally, we examined oxidation of Met, Trp and dioxidation of Trp, along with deamidation (Asn, Gln), pyro-Glu(E), pyroGlu(Q) and carbamylation of the N-terminus and Lys. Digestion efficiency was assessed using the percentage of canonical tryptic peptides, peptides with a missed cleavage and semi-tryptic cleavage. A local FDR of 5% was used at the peptide level to assess these parameters. In ProteinPilot, a 5% local FDR setting is more stringent than a 1% global FDR setting. A two-tailed student’s t-test was used to assess significance, with the threshold set at
