Article pubs.acs.org/jpr
Resolubilization of Precipitated Intact Membrane Proteins with Cold Formic Acid for Analysis by Mass Spectrometry Alan A. Doucette,*,† Douglas B. Vieira,† Dennis J. Orton,‡ and Mark J. Wall† †
Department of Chemistry and ‡Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada S Supporting Information *
ABSTRACT: Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at −20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins. KEYWORDS: acetone, chloroform, precipitation, membrane protein, formic acid, top down MS, GELFrEE
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protein complexation18 are also being realized for intact membrane proteins. MS detection of intact proteins relies on maintaining sufficient analyte concentration and purity through the various stages of sample preparation. Though nonionic detergents will solubilize some membrane proteins or protein complexes,18 as demonstrated by Masuda, SDS is the preferred surfactant for maximal recovery of membrane proteins.10 SDS also maintains solubility during proteome prefractionation, as seen with SDS PAGE or GELFrEE separation.19 Owing to incompatibilities with LC20 and ESI-MS,21 SDS must be depleted prior to analysis.22 The depletion step is a potential source of analyte loss and raises the question of how to maintain protein solubility in the absence of SDS. With bottom up approaches, it is known that trypsin activity is maintained in up to 0.1% SDS, allowing SDS to be depleted following digestion.23 In the FASP and related approaches, MS-compatible additives, such as urea or more recently, sodium deoxycholate,24,25 have been used to enhance solubilization of protein. However, without the benefit of enzyme digestion to generate soluble peptides, maintaining solubility of intact proteins poses additional challenges. Among the many strategies for SDS depletion at the level of intact proteins, solvent-based protein precipitation is a favored approach. Wessel and Flügge’s approach combines chloroform,
INTRODUCTION
Mass spectrometry (MS) is indispensable to the highthroughput characterization of proteins. MS analysis of membrane proteins has garnered significant attention given their importance as targets for new drugs.1,2 However, these proteins are underrepresented in traditional MS detection platforms, owing mainly to difficulties in solubilizing hydrophobic components in MS-compatible solvent systems. Our objectives are to expand the capacity of electrospray ionizationmass spectrometry (ESI-MS) for analysis of intact membrane proteins. Considering bottom up approaches, numerous strategies are suited to detect peptides derived from membrane proteins, including the FASP approach,3 the use of in-gel digestion,4 CNBr,5 proteinase K,6 sodium dodecyl sulfate (SDS)-assisted digestion,7,8 or employing removable detergents including perfluorooctanoic acid,9 acid cleavable8 and phase transfer surfactants.10 Despite obvious progress, bottom up approaches are not without limitations. Most notably, protein sequence coverage is rarely complete. The colocalization of protein modifications is also lost when a protein is digested.11 Top down MS approaches characterize proteins at the intact level. With ESI-MS, the generation of a charge envelope allows for accurate molecular weight determination of membrane proteins,12,13 with subsequent full protein characterization by MS/MS.14,15 Ligand interactions,16 conformation studies,17 and © XXXX American Chemical Society
Received: August 18, 2014
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dx.doi.org/10.1021/pr500864a | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
methanol and water (CMW) to precipitate protein.26 Our group and others have also shown acetone precipitation to deplete SDS ahead of MS.22,27 The recovery of hydrophilic (water-soluble) proteins through acetone precipitation is high (80−100%).28 Though membrane protein recovery has not been fully evaluated, it has been suggested that acetone precipitation of hydrophobic proteins may be compromised.29,30 In CMW, extremely hydrophobic proteins may partition into the chloroform layer.31,32 Protein recovery through CMW has been debated, with reports ranging from below 50%27,33 to near 100%.34 These variations may be attributed to inconsistencies in collecting the protein pellet (i.e., accidental loss during pipetting).35 Following precipitation, protein resolubilization is perhaps of even greater concern as a source of sample loss. Realizing the potential of organic solvents or MS-compatible surfactants to facilitate resolubilization, water alone has also been employed to solubilize precipitated membrane proteins for top down MS.36,37 In water, the solubility of the more hydrophobic components will be compromised, which is a concern as top down proteomics moves toward comprehensive and quantitative analysis.38 A high concentration of formic acid (50−90%) is perhaps the most effective solvent for complete solubilization of hydrophobic membrane proteins.39 Unfortunately, formic acid is known to covalently modify proteins;40,41 the added carbonyl group yielding multiple +28 Da adducts in an MS spectrum. Covalent modifications lower MS signal intensity, broaden chromatographic peaks, and also prevent characterization of intrinsic protein formylation. Whitelegge recommends that protein exposure to formic acid be limited (
