An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.     

Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane

Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.     

Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins

Hydrophobic membrane proteins often have complex functions and are thus of great interest. However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation. We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis. CNBr selectively cleaves methionine residues. But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces. To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel. The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied. We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature. This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples. The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.     

Standardisation of rapid in-gel digestion by mass spectrometry

In-gel digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple in-gel digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in-gel digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin in-gel uptake is increased and in-gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.     

A combination method of chemical with enzyme reactions for identification of membrane proteins

A simple method for effective analysis of various proteins has been developed, including membrane proteins, with LC-MS/MS, using CNBr and acetic acid cleavage in one reaction for the digestion of both the M/ and /D/ positions within the target proteins. This dual chemical reaction has been compared with traditional CNBr or an acid cleavage method using a rat kidney membrane fraction and it showed an advantage of the dual reaction with respect to a high number of peptides detected and a high protein recovery. Furthermore, when this dual chemical reaction was combined with trypsin digestion, the number of proteins surprisingly increased approximately 3.0 times more than in the cases with the trypsin digestion only. It was also 1.9 times more than in cases dealing with Tube-Gel trypsin digestion, which is one of the most efficient digestion methods. In addition, it was shown that this dual chemical reaction could be applied to an in-gel digestion. Using the combination of the chemical and enzyme reaction, 172 proteins including 95 membrane proteins were identified. This indicated that this method is one of the efficient systems in single MS/MS analysis. In particular, many membrane proteins identified in this study were detected by a new combination, but not by a traditional trypsin digestion method.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.     

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.     

Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS

Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.     

Characterization of the Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative Standard using SDS-PAGE shotgun proteomics

Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI.     

High recovery HPLC separation of lipid rafts for membrane proteome analysis

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components. Human brain tissue lipid raft protein preparations have been used as a model system to develop RP-HPLC conditions that are effective for protein fractionation, and are compatible with downstream proteomic analytical workflows. By the use of an appropriate RP column material and operational conditions, human brain membrane raft proteins were successfully resolved by RP-HPLC and some of the protein components, including specific integral membrane proteins, identified by downstream SDS-PAGE combined with in-gel digestion, or in-solution digestion and LC-MS/MS analysis of tryptic fragments. Using the described method, total protein recovery was high, and the repeatability of the separation maintained after repeated injections of membrane raft preparations.     

Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.     

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.     

Protease inhibitors as possible pitfalls in proteomic analyses of complex biological samples

Sample preparation, especially protein and peptide fractionation prior to identification by mass spectrometry (MS), is typically applied to reduce sample complexity. The second key element in this process is proteolytic digestion, which is performed most often with trypsin. Optimization of this step is an important factor in order to achieve both speed and better performance of proteomic analysis, and tryptic digestion prior to the MS analysis has been a topic of many studies. To date, only a few studies have paid attention to the negative interaction between the proteolytic enzyme and sample components, and sample losses caused by these interactions. In this study, we demonstrated impaired activity after "in solution" tryptic digestion of plasma proteins caused by a potent trypsin inhibitor family, inter-alpha inhibitor proteins. Sample boiling followed by gel electrophoretic separation and "in-gel" digestion drastically improved both the number of identified proteins and the sequence coverage in subsequent LC-ESI-MS/MS. The present investigations show that a thorough validation is necessary when "in solution" digestion followed by LC-MS analysis of complex biological samples is performed. The parallel use of two or more different mass spectrometers can also yield additional information and contribute to further method validation.     

Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.     

Mass spectrometric analysis of cyanogen bromide fragments of integral membrane proteins at the picomole level: application to rhodopsin

Advances in time-of-flight mass spectrometry allow unit mass resolution of proteins and peptides up to about 6000 Da molecular weight. Identification of larger proteins and study of their posttranslational or experimental modifications by mass analysis is greatly enhanced by cleavage into smaller fragments. Most membrane proteins are difficult to mass analyze because of their high hydrophobicity, typical expression in low quantities, and because the detergents commonly used for solubilization may be deleterious to mass analysis. Cleavage with cyanogen bromide is beneficial for analysis of membrane proteins since the methionine cleavage sites are typically located in hydrophobic domains and cleavage at these points reduces the size of the hydrophobic fragments. Cyanogen bromide also gives high cleavage yields and introduces only volatile contaminants. Even after cleavage membrane proteins often contain fragments that are difficult to chromatograph. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is capable of analyzing complex mixtures without chromatography. We present a MALDI MS method that quickly and reliably identifies the cyanogen bromide fragments and posttranslational modifications of reduced and alkylated bovine rhodopsin from as little as 30 pmol of rhodopsin in detergent-solubilized retinal rod disk membranes, using 1-5 pmol of digest per sample. The amino acid sequences of some of the peptides in the digest were confirmed by post source decomposition MS analysis of the same samples. The method appears to be general and applicable to the analysis of membrane proteins and the protein composition of membrane preparations.     

Preparation and application of a partially degradable gel in mass spectrometry-based proteomic analysis

In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery method by means of a simple replacement of bis-acrylamide (BA) with bis-acrylylcystamine (BAC). Concretely, the protein sample solubilized using high concentrations of sodium dodecyl sulfate (SDS) and urea were directly entrapped and immobilized into BAC-crosslinked gel by vacuum-dried gel absorption followed by fixation treatment. After removal of SDS and urea by repeated washing, the proteins were subjected to in-gel digestion and the gel was reductively treated. The tryptic peptides were recovered from the partial degradation of the gel and analyzed afterwards by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). Compared with conventional BA-crosslinked gel method, this new method increased the numbers of identified proteins and unique peptides by 20.2% and 20.4%, respectively. The further statistical analysis demonstrated that the method improved the recovery of tryptic peptides particularly larger and/or hydrophobic peptides, thereby significantly facilitating protein identification. Thus, the newly developed method is a promising alternative for BA-crosslinked gel-based shotgun workflows and has potential application in the related fields of protein chemistry and proteomics.