A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides

Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction.     

膜整合蛋白质的“鸟枪法”蛋白质组学分析策略

疾病状态下生物膜表面蛋白质分子标记的表达量和修饰状态会发生改变。但由于其低丰度和不易溶解等特性,制约了膜蛋白质组学的研究,同时也制约了相关药物靶标的设计。近年来,为克服这些困难,学者们提出了"鸟枪法"的膜蛋白质组学研究策略。基于此,本文论述了"鸟枪法"的蛋白质组学分析的基本过程及其后续的部分改进工作。随着新的策略不断被采用,更多膜蛋白质的拓扑学特征和功能的相关研究一定会走上新的台阶。     

A method for the comprehensive proteomic analysis of membrane proteins

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.     

A proteomic approach for the identification of bismuth-binding proteins in Helicobacter pylori

Helicobacter pylori is a major human pathogen that can cause peptic ulcers and chronic gastritis. Bismuth-based triple or quadruple therapies are commonly recommended for the treatment of H. pylori infections. However, the molecular mechanisms underlying treatment with bismuth are currently not fully understood. We have conducted a detailed comparative proteomic analysis of H. pylori cells both before and after treatment with colloidal bismuth subcitrate (CBS). Eight proteins were found to be significantly upregulated or downregulated in the presence of CBS (20 μg mL⁻¹). Bismuth-induced oxidative stress was confirmed by detecting higher levels of lipid hydroperoxide (approximately 1.8 times) and hemin (approximately 3.4 times), in whole cell extracts of bismuth-treated H. pylori cells, compared with those from untreated cells. The presence of bismuth also led to an approximately eightfold decrease in cellular protease activities. Using immobilized-bismuth affinity chromatography, we isolated and subsequently identified seven bismuth-binding proteins from H. pylori cell extracts. The intracellular levels of four of these proteins (HspA, HspB, NapA and TsaA) were influenced by the addition of CBS, which strongly suggests that they interact directly with bismuth. The other bismuth-interacting proteins identified were two enzymes (fumarase and the urease subunit UreB), and a translational factor (Ef-Tu). Our data suggest that the inhibition of proteases, modulation of cellular oxidative stress and interference with nickel homeostasis may be key processes underlying the molecular mechanism of bismuth’s actions against H. pylori. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fast and accurate identification of semi-tryptic peptides in shotgun proteomics

MOTIVATION: One of the major problems in shotgun proteomics is the low peptide coverage when analyzing complex protein samples. Identifying more peptides, e.g. non-tryptic peptides, may increase the peptide coverage and improve protein identification and/or quantification that are based on the peptide identification results. Searching for all potential non-tryptic peptides is, however, time consuming for shotgun proteomics data from complex samples, and poses a challenge for a routine data analysis. RESULTS: We hypothesize that non-tryptic peptides are mainly created from the truncation of regular tryptic peptides before separation. We introduce the notion of truncatability of a tryptic peptide, i.e. the probability of the peptide to be identified in its truncated form, and build a predictor to estimate a peptide's truncatability from its sequence. We show that our predictions achieve useful accuracy, with the area under the ROC curve from 76% to 87%, and can be used to filter the sequence database for identifying truncated peptides. After filtering, only a limited number of tryptic peptides with the highest truncatability are retained for non-tryptic peptide searching. By applying this method to identification of semi-tryptic peptides, we show that a significant number of such peptides can be identified within a searching time comparable to that of tryptic peptide identification.     

A proteomic approach for the identification of cell-surface proteins shed by metalloproteases

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.     

A method for preparing proteins and peptides for microsequencing

We present simple methods for the generation and separation of peptides suitable for automated amino acid sequencing using Edman chemistry. We describein-situ CNBr cleavage of proteins electrophoretically separated in polyacrylamide gels. The generated peptides are separated by gel electrophoresis and blotted onto polyvinylidene diflouride membrane. Membranebound peptides are detected by Coomassie Blue staining and directly applied to an automated sequencer.     

Enhanced detection method for corneal protein identification using shotgun proteomics

Background  

The cornea is a specialized transparent connective tissue responsible for the majority of light refraction and image focus for the retina. There are three main layers of the cornea: the epithelium that is exposed and acts as a protective barrier for the eye, the center stroma consisting of parallel collagen fibrils that refract light, and the endothelium that is responsible for hydration of the cornea from the aqueous humor. Normal cornea is an immunologically privileged tissue devoid of blood vessels, but injury can produce a loss of these conditions causing invasion of other processes that degrade the homeostatic properties resulting in a decrease in the amount of light refracted onto the retina. Determining a measure and drift of phenotypic cornea state from normal to an injured or diseased state requires knowledge of the existing protein signature within the tissue. In the study of corneal proteins, proteomics procedures have typically involved the pulverization of the entire cornea prior to analysis. Separation of the epithelium and endothelium from the core stroma and performing separate shotgun proteomics using liquid chromatography/mass spectrometry results in identification of many more proteins than previously employed methods using complete pulverized cornea.     

Strategies for shotgun identification of integral membrane proteins by tandem mass spectrometry

Integral membrane proteins (IMPs) are difficult to identify, mainly for two reasons: the hydrophobicity of IMPs and their low abundance. Sample preparation is a key component in the large-scale identification of IMPs. In this review, we survey strategies for shotgun identification of IMPs by MS/MS. We will discuss enrichment, solubilization, separation, and digestion of IMPs, and data analysis for membrane proteomics.     

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry was employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu⁴⁺-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore™ analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.     

DBParser: web-based software for shotgun proteomic data analyses

We describe a web-based program called 'DBParser' for rapidly culling, merging, and comparing sequence search engine results from multiple LC-MS/MS peptide analyses. DBParser employs the principle of parsimony to consolidate redundant protein assignments and derive the most concise set of proteins consistent with all of the assigned peptide sequences observed in an experiment or series of experiments. The resulting reports summarize peptide and protein identifications from multidimensional experiments that may contain a single data set or combine data from a group of data sets, all related to a single analytical sample. Additionally, the results of multiple experiments, each of which may contain several data sets, can be compared in reports that identify features that are common or different. DBParser actively links to the primary mass spectral data and to public online databases such as NCBI, GO, and Swiss-Prot in order to structure contextually specific reports for biologists and biochemists.     

Monte carlo simulation-based algorithms for analysis of shotgun proteomic data

Two new statistical models based on Monte Carlo Simulation (MCS) have been developed to score peptide matches in shotgun proteomic data and incorporated in a database search program, MassMatrix (www.massmatrix.net). The first model evaluates peptide matches based on the total abundance of matched peaks in the experimental spectra. The second model evaluates amino acid residue tags within MS/MS spectra. The two models provide complementary scores for peptide matches that result in higher confidence in peptide identification when significant scores are returned from both models. The MCS-based models use a variance reduction technique that improves estimation precision. Due to the high computational expense of MCS-based models, peptide matches were prefiltered by other statistical models before further evaluation by the MCS-based models. Receiver operating characteristic analysis of the data sets confirmed that MCS-based models improved the overall performance of the MassMatrix search software, especially for low-mass accuracy data sets.     

A method for the isolation and identification of pyridoxal phosphate in proteins

A method for the isolation and identification of covalently bound pyridoxal phosphate (PLP) contained in some enzymatic proteins is presented. The method involves acid hydrolysis of the protein in the presence of phenylhydrazine, separation of the adduct by elution from Sep-Pak C18 cartridges, isolation by HPLC, and either direct analysis by mass spectrometry with direct electron impact or conversion into trimethylsilyl derivatives followed by gas chromatography-mass spectrometry. Under the prescribed conditions of hydrolysis, PLP forms its phenylhydrazone and is released from the protein and hydrolyzed to the phenylhydrazone of pyridoxal, which shows a typical fragmentation in direct electron impact and in gas chromatography-mass spectrometry after silylation. The yield in phenylhydrazone of pyridoxal is on the order of 50% (+/- 5% SE, n = 15) when PLP is added to 10 mg of protein in amounts ranging from 20 to 40 nmol. Analysis of pig plasma benzylamine oxidase by this procedure confirms the presence of covalently bound pyridoxal phosphate in this enzyme.     

A general method for the isolation of labelled peptides from affinity-labelled proteins

A general method for specific isolation of ligand-bound peptides from affinity-labelled proteins is described. The method makes use of the affinity of the native protein to the ligand that was used for labelling of the same protein. Thus the labelled peptide can be isolated in one step in a very rapid procedure. The method was applied successfully to peptides from an antibody and from ribonuclease.     

Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses.     

Dynamic spectrum quality assessment and iterative computational analysis of shotgun proteomic data: toward more efficient identification of post-translational modifications, sequence polymorphisms, and novel peptides

In mass spectrometry-based proteomics, frequently hundreds of thousands of MS/MS spectra are collected in a single experiment. Of these, a relatively small fraction is confidently assigned to peptide sequences, whereas the majority of the spectra are not further analyzed. Spectra are not assigned to peptides for diverse reasons. These include deficiencies of the scoring schemes implemented in the database search tools, sequence variations (e.g. single nucleotide polymorphisms) or omissions in the database searched, post-translational or chemical modifications of the peptide analyzed, or the observation of sequences that are not anticipated from the genomic sequence (e.g. splice forms, somatic rearrangement, and processed proteins). To increase the amount of information that can be extracted from proteomic MS/MS datasets we developed a robust method that detects high quality spectra within the fraction of spectra unassigned by conventional sequence database searching and computes a quality score for each spectrum. We also demonstrate that iterative search strategies applied to such detected unassigned high quality spectra significantly increase the number of spectra that can be assigned from datasets and that biologically interesting new insights can be gained from existing data.     

A new acid hydrolysis method for determining tryptophan in peptides and proteins

Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.