Mass spectrometry-based proteomics combined with bioinformatic tools for bacterial classification

Timely classification and identification of bacteria is of vital importance in many areas of public health. We present a mass spectrometry (MS)-based proteomics approach for bacterial classification. In this method, a bacterial proteome database is derived from all potential protein coding open reading frames (ORFs) found in 170 fully sequenced bacterial genomes. Amino acid sequences of tryptic peptides obtained by LC-ESI MS/MS analysis of the digest of bacterial cell extracts are assigned to individual bacterial proteomes in the database. Phylogenetic profiles of these peptides are used to create a matrix of sequence-to-bacterium assignments. These matrixes, viewed as specific assignment bitmaps, are analyzed using statistical tools to reveal the relatedness between a test bacterial sample and the microorganism database. It is shown that, if a sufficient amount of sequence information is obtained from the MS/MS experiments, a bacterial sample can be classified to a strain level by using this proteomics method, leading to its positive identification.     

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.     

Mass spectrometry-based glycoproteomic approach involving lysine derivatization for structural characterization of recombinant human erythropoietin

Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

A new protein from human plasma lipoproteins: isolation and partial characterization of apolipoprotein G]

A new apolipoprotein has been identified in VHDL1 and in HDL. This protein is immunologically distinct from already isolated apoproteins. It was isolated by column chromatography on hydroxylapatite. In polyacrylamide gel electrophoresis, its mobility is very close to that of apo D. The amino acid composition differs from those of the well characterized polypeptides of the human plasma lipoproteins. It contains glucosamine. The apparent molecular weight is 72 000 +/- 2 000 in the presence and absence of reducing agent. According to the ABCDEF nomenclature, this protein can be named apolipoprotein G (apo G). It is present in a lipoprotein distinct from the lipoproteins A and D among the VHDL1 : this new lipoprotein can be named lipoprotein G (LPG).     

Experimental and analytical tools for studying the human microbiome

The human microbiome substantially affects many aspects of human physiology, including metabolism, drug interactions and numerous diseases. This realization, coupled with ever-improving nucleotide sequencing technology, has precipitated the collection of diverse data sets that profile the microbiome. In the past 2 years, studies have begun to include sufficient numbers of subjects to provide the power to associate these microbiome features with clinical states using advanced algorithms, increasing the use of microbiome studies both individually and collectively. Here we discuss tools and strategies for microbiome studies, from primer selection to bioinformatics analysis.     

Mass spectrometry-based footprinting of protein–protein interactions

We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein–protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein–protein interactions that are less amenable for crystallographic or NMR analysis.     

Mass spectrometry-based plasma proteomics: state of the art and future outlook

Mass spectrometry-based plasma proteomics is a field where intense research has been performed during the last decade. Being closely linked to biomarker discovery, the field has received a fair amount of criticism, mostly due to the low number of novel biomarkers reaching the clinic. However, plasma proteomics is under gradual development with improvements on fractionation methods, mass spectrometry instrumentation and analytical approaches. These recent developments have contributed to the revival of plasma proteomics. The goal of this review is to summarize these advances, focusing in particular on fractionation methods, both for targeted and global mass spectrometry-based plasma analysis.     

Mass spectrometry-based analyses for identifying and characterizing S-nitrosylation of protein tyrosine phosphatases

All members in the protein tyrosine phosphatase (PTP) family of enzymes contain an invariant Cys residue which is absolutely indispensable for catalysis. Due to the unique microenvironment surrounding the active center of PTPs, this Cys residue exhibits an unusually low pKa characteristic, thus being highly susceptible to oxidation or S-nitrosylation. While oxidation-dependent regulation of PTP activity has been extensively examined, the molecular details and biological consequences of PTP S-nitrosylation remain unexplored. We hypothesized that the catalytic Cys residue is targeted by proximal nitric oxide (NO) and its derivatives collectively termed reactive nitrogen species (RNS), leading to nitrosothiol formation concomitant with reversible inactivation of PTPs. To test this hypothesis, we have developed novel strategies to examine the redox status of Cys residues of purified PTP1B that was exposed to NO donor S-Nitroso-N-penicillamine (SNAP). A gel-based method in conjunction with mass spectrometry (MS) analysis revealed that the catalytic Cys215 of PTP1B was reversibly modified when PTP1B was briefly treated with SNAP. In order to further identify the exact mode of NO-induced modification, we employed an online LC-ESI-MS/MS analysis incorporating a mass difference-based, data-dependent acquisition function that effectively mapped the S-nitrosylated Cys residues. Our results demonstrated that treating PTP1B with SNAP led to S-nitrosothiol formation of the catalytic Cys215. Interestingly, SNAP-induced modifications were strictly reversible as highly oxidized Cys derivatives (Cys-SO(2)H or Cys-SO(3)H) were not identified by MS analyses. Thus, the methods introduced in this study provide direct evidence to prove the direct link between S-nitrosylation of the catalytic Cys residue and reversible inactivation of PTPs.     

Mass spectrometry-based proteomics for systems biology

Mass spectrometry (MS)-based proteomics has significantly contributed to the development of systems biology, a new paradigm for the life sciences in which biological processes are addressed in terms of dynamic networks of interacting molecules. Because of its advanced analytical capabilities, MS-based proteomics has been used extensively to identify the components of biological systems, and it is the method of choice to consistently quantify the effects of network perturbation in time and space. Herein, we review recent contributions of MS to systems biology and discuss several examples that illustrate the importance of mass spectrometry to elucidate the components and interactions of molecular networks.     

Mass spectrometry-based study of the plasma proteome in a mouse intestinal tumor model

Early detection of cancer can greatly improve prognosis. Identification of proteins or peptides in the circulation, at different stages of cancer, would greatly enhance treatment decisions. Mass spectrometry (MS) is emerging as a powerful tool to identify proteins from complex mixtures such as plasma that may help identify novel sets of markers that may be associated with the presence of tumors. To examine this feature we have used a genetically modified mouse model, Apc(Min), which develops intestinal tumors with 100% penetrance. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified total plasma proteome (TPP) and plasma glycoproteome (PGP) profiles in tumor-bearing mice. Principal component analysis (PCA) and agglomerative hierarchial clustering analysis revealed that these protein profiles can be used to distinguish between tumor-bearing Apc(Min) and wild-type control mice. Leave-one-out cross-validation analysis established that global TPP and global PGP profiles can be used to correctly predict tumor-bearing animals in 17/19 (89%) and 19/19 (100%) of cases, respectively. Furthermore, leave-one-out cross-validation analysis confirmed that the significant differentially expressed proteins from both the TPP and the PGP were able to correctly predict tumor-bearing animals in 19/19 (100%) of cases. A subset of these proteins was independently validated by antibody microarrays using detection by two color rolling circle amplification (TC-RCA). Analysis of the significant differentially expressed proteins indicated that some might derive from the stroma or the host response. These studies suggest that mass spectrometry-based approaches to examine the plasma proteome may prove to be a valuable method for determining the presence of intestinal tumors.     

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.     

Mass spectrometry-based proteomic studies of human anaplastic large cell lymphoma

Malignant lymphomas are a diverse group of malignant neoplasms that arise as a result of a complex interplay of multiple factors including genetic aberrations, immunosuppression, and exposure to noxious agents such as ionizing radiation and chemical agents. Anaplastic large cell lymphoma (ALCL) is an aggressive T-lineage lymphoma harboring chromosomal translocations involving the anaplastic lymphoma kinase (ALK) tyrosine kinase. The most common translocation in ALCL is the t(2;5)(p23;q35). This results in the formation of a chimeric fusion kinase, nucleophosmin/ALK. Nucleophosmin/ALK activates numerous downstream signaling pathways resulting in enhanced survival and proliferation. Using a variety of mass spectrometry-driven proteomic strategies, we have studied several aspects of the ALCL proteome. In this review, we provide a summary of mass spectrometry-based proteomic studies that expands the current understanding of the molecular pathogenesis of ALCL and provides the basis for the identification of biomarkers and targets for novel therapeutic agents.     

Mass spectrometry-based detection and quantification of plasma glycoproteins using selective reaction monitoring

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.