Effects of common surfactants on protein digestion and matrix‐assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two‐layer sample preparation

Abstract

While surfactants are commonly used in preparing protein samples, their presence in a protein sample can potentially affect the enzymatic digestion process and the subsequent analysis of the resulting peptides by mass spectrometry. The extent of the tolerance of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to surfactant interference in peptide analysis is very much dependent on the matrix/sample preparation method. In this work the effects of four commonly used surfactants, namely n-octyl glucoside (OG), Triton X-100 (TX-100), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS), for biological sample preparation on trypsin digestion and MALDI-MS of the resulting digest are examined in detail within the context of using a two-layer method for MALDI matrix/sample preparation. Non-ionic and mild surfactants, such as OG, TX-100 or CHAPS, are found to have no significant effect on trypsin digestion with surfactant concentrations up to 1%. However, TX-100 and CHAPS interfere with the subsequent peptide analysis by MALDI-MS and should be removed prior to peptide analysis. OG is an MS-friendly surfactant and no effect is observed for MALDI peptide analysis. The effect of SDS on trypsin digestion in terms of the number of peptides generated and the overall protein sequence coverage by these peptides is found to be protein dependent. The use of SDS to solubilize hydrophobic membrane proteins, followed by trypsin digestion in the presence of 0.1% SDS, results in a peptide mixture that can be analyzed directly by MALDI-MS. These peptides are shown to provide better sequence coverage compared with those obtained without the use of SDS in the case of bacteriorhodopsin, a very hydrophobic transmembrane protein. This work illustrates that MALDI-MS with the two-layer sample preparation method can be used for direct analysis of protein digests with no or minimum sample cleanup after proteins are digested in a solution containing surfactants.

Interactions between sodium dodecyl sulfate micelles and peptides during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of proteolytic digests

“…However, at very high concentrations (Ͼ1% wt/vol) of SDS the formation of crystals was difficult, it took longer for the samples to dry and excess sodium in the sample led to a deterioration of spectral quality. These trends are similar°to°those°reported°by°Zhang°and°Li° [29].…”

“…Similarly, BSA (66 kDa) digested in bicarbonate buffer resulted in an increase in sequence coverage from 28 to 45% when prepared with SDS near its CMC. While Zhang and Li have found that the presence of lower concentrations of SDS (Ͻ1%) does not affect tryptic digestion of proteins, they did not observe any trends relating to sequence coverage°as°a°function°of°SDS°concentration° [29].°We attribute the differences between these two findings to the sample preparation steps. SDS is added in the present study after tryptic digestion and the SDSpeptide mixture is vortexed thoroughly to ensure optimal peptide-micelle interactions.…”

“…Li and coworkers have shown that various aspects like on-probe washing, choice of matrix, solvent, protein concentration, and sample preparation method affect the abundance of MALDI signals for SDS containing samples [28]. Using the two-layer sample preparation method, they established that 1% SDS can be tolerated in peptide mapping experiments and 2% SDS can be tolerated in the analysis of proteins [29].…”

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

“…The most common detergents are CHAPS and IGEPAL CA‐630. Detergents have to be removed before downstream LC‐MS analysis as they significantly influence peptide separation on the LC column53, 54 or interfere with ionization of analytes 55, 56. Therefore, thorough and repeated washing of IP samples is necessary.…”

Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography–Tandem Mass Spectrometry

“…Here, we compared the efficiencies of urea, SDC, and OG for generating high yields of proteotypic peptides of 13 UGT isoforms in four biologic specimens. The efficiencies of proteolysis by trypsin in the presence of these denaturing agents are generally higher than those in the absence of the denaturant (Katayama et al, 2001(Katayama et al, , 2004Zhang and Li, 2004;Zhou et al, 2006;Chen et al, 2007;Masuda et al, 2008;Proc et al, 2010;Balogh et al, 2012). As done by others, the best practice would be investigating multiple peptides per protein when available (Harbourt et al, 2012;Ohtsuki et al, 2012;Schaefer et al, 2012;Fallon et al, 2013b;Achour et al, 2014).…”