Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

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.

[1]  Paul M Thomas,et al.  Large-scale Top-down Proteomics of the Human Proteome: Membrane Proteins, Mitochondria, and Senescence* , 2013, Molecular & Cellular Proteomics.

[2]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[3]  P. van Gelder,et al.  Structure and function of bacterial outer membrane proteins: barrels in a nutshell , 2000, Molecular microbiology.

[4]  A. Doucette,et al.  Dual LC-MS platform for high-throughput proteome analysis. , 2013, Journal of proteome research.

[5]  A. Doucette,et al.  Maximizing recovery of water-soluble proteins through acetone precipitation. , 2013, Analytica chimica acta.

[6]  R. Hettich,et al.  Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization. , 2012, Journal of proteome research.

[7]  Liang Li,et al.  Off-line two-dimensional liquid chromatography with maximized sample loading to reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry for shotgun proteome analysis. , 2009, Analytical chemistry.

[8]  S. Kędracka-Krok,et al.  Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis , 2010, Electrophoresis.

[9]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[10]  Evaluation of enzymatic digestion and liquid chromatography‐mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin , 2002, Electrophoresis.

[11]  J. C. Tran,et al.  Gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation. , 2008, Analytical chemistry.

[12]  M. Mann,et al.  Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells , 2014, Nature Methods.

[13]  Masaru Tomita,et al.  Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. , 2008, Journal of proteome research.

[14]  J. C. Tran,et al.  Multiplexed size separation of intact proteins in solution phase for mass spectrometry. , 2009, Analytical chemistry.

[15]  Liang Li,et al.  Comparison of surfactant-assisted shotgun methods using acid-labile surfactants and sodium dodecyl sulfate for membrane proteome analysis. , 2011, Analytica chimica acta.

[16]  M. Miyagi,et al.  Perfluorooctanoic Acid for Shotgun Proteomics , 2010, PloS one.

[17]  J. Yates,et al.  A model for random sampling and estimation of relative protein abundance in shotgun proteomics. , 2004, Analytical chemistry.

[18]  H. Kawasaki,et al.  Separation of peptides dissolved in a sodium dodecyl sulfate solution by reversed-phase liquid chromatography: removal of sodium dodecyl sulfate from peptides using an ion-exchange precolumn. , 1990, Analytical biochemistry.

[19]  B. Chait,et al.  Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. , 1992, Science.

[20]  Joseph A. Loo,et al.  Enhanced FASP (eFASP) to Increase Proteome Coverage and Sample Recovery for Quantitative Proteomic Experiments , 2014, Journal of proteome research.

[21]  M. Mann,et al.  In-gel digestion for mass spectrometric characterization of proteins and proteomes , 2006, Nature Protocols.

[22]  Visith Thongboonkerd,et al.  Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. , 2002, Kidney international.

[23]  A. Doucette,et al.  Top-down and bottom-up proteomics of SDS-containing solutions following mass-based separation. , 2010, Journal of proteome research.

[24]  E. Bellei,et al.  Optimizing protein recovery yield from serum samples treated with beads technology , 2011, Electrophoresis.

[25]  K. Blennow,et al.  Removal of sodium dodecyl sulfate from protein samples prior to matrix-assisted laser desorption/ionization mass spectrometry. , 1999, Rapid communications in mass spectrometry : RCM.

[26]  Neil L. Kelleher,et al.  Applying Label-Free Quantitation to Top Down Proteomics , 2014, Analytical chemistry.

[27]  G. Schulz,et al.  High-resolution structure of the OmpA membrane domain. , 2000, Journal of molecular biology.

[28]  Andrew J. Doig,et al.  Properties and identification of human protein drug targets , 2009, Bioinform..

[29]  J. Lee,et al.  Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  D. Armstrong,et al.  Mechanism of signal suppression by anionic surfactants in capillary electrophoresis-electrospray ionization mass spectrometry. , 1996, Analytical chemistry.

[31]  J. Pettegrew,et al.  Alzheimer's β‐Amyloid Protein Is Covalently Modified when Dissolved in Formic Acid , 1990, Journal of neurochemistry.

[32]  J. Yates,et al.  A method for the comprehensive proteomic analysis of membrane proteins , 2003, Nature Biotechnology.

[33]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[34]  M. Mann,et al.  Universal sample preparation method for proteome analysis , 2009, Nature Methods.

[35]  A. Berry,et al.  A reactive, surface cysteine residue of the class-II fructose-1,6-bisphosphate aldolase of Escherichia coli revealed by electrospray ionisation mass spectrometry. , 1995, European journal of biochemistry.

[36]  Xiaoning Lu,et al.  Tube-Gel Digestion , 2005, Molecular & Cellular Proteomics.

[37]  G. Ghiggeri,et al.  Combinatorial peptide ligand libraries for urine proteome analysis: Investigation of different elution systems , 2009, Electrophoresis.

[38]  John Davey,et al.  G-Protein-Coupled Receptors: New Approaches to Maximise the Impact of GPCRs in Drug Discovery , 2004, Expert opinion on therapeutic targets.

[39]  B. Chait,et al.  Rapid, sensitive analysis of protein mixtures by mass spectrometry. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Schaller,et al.  Analysis of Hydrophobic Proteins and Peptides by Electrospray Ionization Mass Spectrometry , 1997 .

[41]  Nan Zhang,et al.  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. , 2004, Rapid communications in mass spectrometry : RCM.

[42]  Liang Li,et al.  Microcolumn capture and digestion of proteins combined with mass spectrometry for protein identification. , 2002, Journal of proteome research.

[43]  N. Kelleher,et al.  Decoding protein modifications using top-down mass spectrometry , 2007, Nature Methods.

[44]  L. Brown,et al.  Hydrogen exchange mass spectrometry of bacteriorhodopsin reveals light-induced changes in the structural dynamics of a biomolecular machine. , 2011, Journal of the American Chemical Society.

[45]  D. Wessel,et al.  A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. , 1984, Analytical biochemistry.

[46]  Carol V Robinson,et al.  Mass spectrometry of intact membrane protein complexes , 2013, Nature Protocols.

[47]  F. Halgand,et al.  Top-down mass spectrometry of integral membrane proteins , 2006, Expert review of proteomics.

[48]  J. Sunner,et al.  Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry. , 1999, Analytical biochemistry.

[49]  Paul M Thomas,et al.  Top down proteomics of human membrane proteins from enriched mitochondrial fractions. , 2013, Analytical chemistry.

[50]  J. Whitelegge.  Tandem mass spectrometry of integral membrane proteins for top-down proteomics , 2005 .

[51]  Carol V. Robinson,et al.  Dynamic protein ligand interactions – insights from MS , 2014, The FEBS journal.

[52]  J. Whitelegge,et al.  Increased coverage in the transmembrane domain with activated-ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins. , 2007, Journal of proteome research.