Mass processing--an improved technique for protein identification with mass spectrometry data.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis following tryptic digestion of polyacrylamide gel pieces is a common technique used to identify proteins. This approach is rapid, sensitive, and user friendly, and is becoming widely available to scientists in a variety of biological fields. Here we introduce a simple and effective strategy called "mass processing" where the list of masses generated from a mass spectrometer undergoes two stages of data reduction before identification. Mass processing improves the ability to identify in-gel tryptic-digested proteins by reducing the number of nonsample masses submitted to protein identification database search engines. Our results demonstrate that mass processing improves the statistical score and rank of putative protein identifications, especially with low-quantity samples, thus increasing the ability to confidently identify proteins with mass spectrometry data.     

Networks for the allosteric control of protein kinases

The allosteric regulation of protein kinases serves as an efficient strategy for molecular communication, event coupling and interconversion between catalytic states. Recent co-crystal structures have revealed novel ways in which kinases control activity and substrate specificity following phosphorylation, dimerization, or binding to regulatory proteins, substrates and scaffolds. In addition, hydrogen exchange coupled with mass spectrometry is emerging as a complementary strategy to probe the solution behavior of kinases; recent results have shown that allosteric regulation may involve transitions in protein motions as well as structural rearrangements.     

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.     

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.     

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.     

Mass spectrometry-based functional proteomics: from molecular machines to protein networks

The study of protein-protein interactions by mass spectrometry is an increasingly important part of post-genomics strategies to understand protein function. A variety of mass spectrometry-based approaches allow characterization of cellular protein assemblies under near-physiological conditions and subsequent assignment of individual proteins to specific molecular machines, pathways and networks, according to an increasing level of organizational complexity. An appropriate analytical strategy can be individually tailored--from an in-depth analysis of single complexes to a large-scale characterization of entire molecular pathways or even an analysis of the molecular organization of entire expressed proteomes. Here we review different options regarding protein-complex purification strategies, mass spectrometry analysis and bioinformatic methods according to the specific question that is being addressed.     

基于谱图库的蛋白质鉴定策略研究进展

基于质谱的蛋白质组学快速发展,蛋白质质谱数据也呈指数式增长。寻找速度快、准确度高以及重复性好的鉴定方法是该领域的一项重要任务。谱图库搜索策略直接比较实验谱图与谱图库中的真实谱图,充分利用了谱图中的丰度、非常规碎裂模式和其他的一些特征,使得搜索更加快速和准确,成为蛋白质组学的主流鉴定方法之一。文中介绍基于谱图库的蛋白质组质谱数据鉴定策略,并针对其中两个关键步骤——谱图库构建方法和谱图库搜索方法进行深入介绍,探讨了谱图库策略的进展和挑战。     

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.     

Developments in mass spectrometry for the analysis of complex protein mixtures.

State-of-the-art proteomics workflows involve multiple interdependent steps: sample preparation, protein-peptide separation, mass spectrometry and data analysis. While improvements in any of these steps can increase the depth and breadth of analysis, advances in mass spectrometry have catalysed many of the most important developments. We discuss common classes of mass analysers and how these analysers are put together to produce some of the most popular mass spectrometry platforms. The capabilities of these platforms determine how they can be used in a variety of common proteomic strategies and, in turn, what types of biological questions can be addressed. Moving forward, powerful new hybrid mass spectrometers and application of emerging types of tandem mass spectrometry promise that our ability to analyse complex mixtures of proteins will continue to advance.     

Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics

Calcineurin is a calcium-calmodulin-dependent serine/threonine specific protein phosphatase operating in key cellular processes governing responses to extracellular cues. Calcineurin is essential for growth at high temperature and virulence of the human fungal pathogen Cryptococcus neoformans but the underlying mechanism is unknown. We performed a mass spectrometry analysis to identify proteins that associate with the calcineurin A catalytic subunit (Cna1) in C. neoformans cells grown under non-stress and high temperature stress conditions. A novel prioritization strategy for mass spectrometry data from immunoprecipitation experiments identified putative substrates and proteins potentially operating with calcineurin in common pathways. Cna1 co-purified with proteins involved in membrane trafficking including the COPI component Sec28 and the COPII component Sec13. The association of Cna1 with Sec28 and Sec13 was confirmed by co-immunoprecipitation. Cna1 exhibited a dramatic change in subcellular localization during high temperature stress from diffuse cytoplasmic to ER-associated puncta and the mother-bud neck and co-localized with Sec28 and Sec13.     

Algorithms for identifying protein cross-links via tandem mass spectrometry.

Cross-linking technology combined with tandem mass spectrometry (MS-MS) is a powerful method that provides a rapid solution to the discovery of protein-protein interactions and protein structures. We studied the problem of detecting cross-linked peptides and cross-linked amino acids from tandem mass spectral data. Our method consists of two steps: the first step finds two protein subsequences whose mass sum equals a given mass measured from the mass spectrometry; and the second step finds the best cross-linked amino acids in these two peptide sequences that are optimally correlated to a given tandem mass spectrum. We designed fast and space-efficient algorithms for these two steps and implemented and tested them on experimental data of cross-linked hemoglobin proteins. An interchain cross-link between two beta subunits was found in two tandem mass spectra. The length of the cross-linker (7.7 A) is very close to the actual distance (8.18 A) obtained from the molecular structure in PDB.     

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.     

Absolute systems biology--measuring dynamics of protein modifications

Mass spectrometry is already established as the method of choice for protein identification. Hitherto, a major limitation of mass spectrometry was the inability to quantitate protein levels. With the development of novel methods, such as the AQUA method by Gerber et al., this hurdle has finally been overcome, unleashing the power of mass spectrometry to study protein dynamics in cells.     

Decoding protein modifications using top-down mass spectrometry

Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.     

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.     

Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry

Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein–protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein–peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium–tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI–TOF instruments and has general utility for the label-free analysis of microarray assays.     

Amino acid sequence of acyl-CoA-binding protein from cow liver.

Acyl-CoA-binding protein from bovine liver was purified with the use of reverse-phase h.p.l.c. in the final step. The complete amino acid sequence was determined by using a combination of gas-phase Edman degradation and electron-impact and fast-atom-bombardment mass spectrometry. The sequence was confirmed by determination of the Mr by plasma-desorption time-of-flight mass spectrometry.     

Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry

Liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here we clarify our preferred methodology by addressing four issues based on observed decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to separate target and decoy database searches; (iii) the theoretical error associated with target-decoy false positive (FP) rate measurements can be estimated; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.     

A data-analytic strategy for protein biomarker discovery: profiling of high-dimensional proteomic data for cancer detection

With recent advances in mass spectrometry techniques, it is now possible to investigate proteins over a wide range of molecular weights in small biological specimens. This advance has generated data-analytic challenges in proteomics, similar to those created by microarray technologies in genetics, namely, discovery of 'signature' protein profiles specific to each pathologic state (e.g. normal vs. cancer) or differential profiles between experimental conditions (e.g. treated by a drug of interest vs. untreated) from high-dimensional data. We propose a data-analytic strategy for discovering protein biomarkers based on such high-dimensional mass spectrometry data. A real biomarker-discovery project on prostate cancer is taken as a concrete example throughout the paper: the project aims to identify proteins in serum that distinguish cancer, benign hyperplasia, and normal states of prostate using the Surface Enhanced Laser Desorption/Ionization (SELDI) technology, a recently developed mass spectrometry technique. Our data-analytic strategy takes properties of the SELDI mass spectrometer into account: the SELDI output of a specimen contains about 48,000 (x, y) points where x is the protein mass divided by the number of charges introduced by ionization and y is the protein intensity of the corresponding mass per charge value, x, in that specimen. Given high coefficients of variation and other characteristics of protein intensity measures (y values), we reduce the measures of protein intensities to a set of binary variables that indicate peaks in the y-axis direction in the nearest neighborhoods of each mass per charge point in the x-axis direction. We then account for a shifting (measurement error) problem of the x-axis in SELDI output. After this pre-analysis processing of data, we combine the binary predictors to generate classification rules for cancer, benign hyperplasia, and normal states of prostate. Our approach is to apply the boosting algorithm to select binary predictors and construct a summary classifier. We empirically evaluate sensitivity and specificity of the resulting summary classifiers with a test dataset that is independent from the training dataset used to construct the summary classifiers. The proposed method performed nearly perfectly in distinguishing cancer and benign hyperplasia from normal. In the classification of cancer vs. benign hyperplasia, however, an appreciable proportion of the benign specimens were classified incorrectly as cancer. We discuss practical issues associated with our proposed approach to the analysis of SELDI output and its application in cancer biomarker discovery.     

Acyl-CoA-binding protein in the rat. Purification, binding characteristics, tissue concentrations and amino acid sequence.

Acyl-CoA-binding protein (ACBP) was purified from rat liver. The Mr was determined as 9932 +/- 10 by mass spectrometry and calculated as 9937.8 from the sequence. The protein binds acyl-CoA esters (C8-C16) with high affinity, but was unable to bind fatty acids. ACBP was found mainly (86%) in the soluble fraction, and the concentration was highest in liver, 5-6 micrograms/mg of soluble protein. The complete primary structure was determined by a combination of gas-phase Edman degradations and mass spectrometry. Extensive use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides. Comparison with the previously determined sequence of bovine acyl-CoA-binding protein revealed a very strong sequence similarity (83%), and all of the differences could be accounted for by single base changes.