Bifunctional glass membrane designed to interface SDS-PAGE separations of proteins with the detection of peptides by mass spectrometry.

We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-PAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.

Interferon-stimulated gene 15 (ISG15) conjugates proteins in dermatomyositis muscle with perifascicular atrophy.

OBJECTIVE: We investigated interferon-stimulated gene 15 (ISG15), a poorly understood ubiquitin-like modifier, and its enzymatic pathway in dermatomyositis (DM), an autoimmune disease primarily involving muscle and skin. METHODS: We generated microarray data measuring transcript abundance for approximately 18,000 genes in each of 113 human muscle biopsy specimens, and studied biopsy specimens and cultured skeletal muscle using immunohistochemistry, immunoblotting proteomics, real-time quantitative polymerase chain reaction, and laser-capture microdissection. RESULTS: Transcripts encoding ISG15-conjugation pathway proteins were markedly upregulated in DM with perifascicular atrophy (DM-PFA) muscle (ISG15 339-fold, HERC5 62-fold, and USP18 68-fold) compared with 99 non-DM samples. Combined analysis with publicly available microarray datasets showed that >50-fold ISG15 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM patients compared with 199 muscle samples from other muscle diseases. Free ISG15 and ISG15-conjugated proteins were only found on immunoblots from DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and ISG15 protein and conjugates. Laser-capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers. INTERPRETATION: A large-scale microarray study of muscle samples demonstrated that among a diverse group of muscle diseases DM was uniquely associated with upregulation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produced a molecular picture highly similar to that seen in human DM muscle. Perifascicular atrophic myofibers in DM were deficient in a number of skeletal muscle proteins including titin.

Using Matrix-Assisted Laser Desorption/Ionization Time of Flight Spectra To Elucidate Species Boundaries by Matching to Translated DNA Databases.

A method has been established to map a bacterial colony to the ever-expanding database of publicly available bacterial genomes by means of matrix-assisted laser desorption/ionization (MALDI) spectra. To accomplish this, spectra are mapped to the predicted masses of ∼65 families of mostly ribosomal proteins. Each of the ∼40 000 bacterial strains in the database receives scores, together with tables listing identified protein sequences and how the highest ranking strains are related to one another. The approach was first confirmed with 16 distinct species of bacteria from the Vibrionales whose genome had been sequenced. Identifications of a few species of bacteria from environmental samples from compost, lakes, and streams in Massachusetts are also reported. Most of these organisms map to known species in the Gammaproteobacteria and Firmicutes. The clades of bacteria deducible from shared ribosomal protein sequences do not always correspond well to named bacterial species. Instead, the identifications made by this methodology indicate groupings of organisms that can readily be distinguished by MALDI-TOF and indicate which polymorphisms in highly conserved proteins demarcate the groupings. Successful identifications highlight organism interrelationships that can be deduced from the available genomes, sorting together genomes into new proposed clades typically consistent with relationships deduced from DNA sequence analysis. In contrast, if for a high-quality spectrum from a fresh colony, no group of related organisms receives high scores, one might infer that no closely related genome has yet been deposited into the database.

Memory T-cell responses to Vibrio cholerae O1 infection.

Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.

Proteomic analysis of Vibrio cholerae in human stool.

An effective vaccine for Vibrio cholerae is not yet available for use in the developing world, where the burden of cholera disease is highest. Characterizing the proteins that are expressed by V. cholerae in the human host environment may provide insight into the pathogenesis of cholera and assist with the development of an improved vaccine. We analyzed the V. cholerae proteins present in the stools of 32 patients with clinical cholera. The V. cholerae outer membrane porin, OmpU, was identified in all of the human stool samples, and many V. cholerae proteins were repeatedly identified in separate patient samples. The majority of V. cholerae proteins identified in human stool are involved in protein synthesis and energy metabolism. A number of proteins involved in the pathogenesis of cholera, including the A and B subunits of cholera toxin and the toxin-coregulated pilus, were identified in human stool. In a subset of stool specimens, we also assessed which in vivo expressed V. cholerae proteins were recognized uniquely by convalescent-phase as opposed to acute-phase serum from cholera patients. We identified a number of these in vivo expressed proteins as immunogenic during human infection. To our knowledge, this is the first characterization of the proteome of a pathogenic bacteria recovered from a natural host.

Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

Scoring methods in MALDI peptide mass fingerprinting: ChemScore, and the ChemApplex program.

No universally accepted score is currently available to determine when a matrix-assisted laser desorption ionization (MALDI) peptide mass fingerprint (PMF) experiment has been successfully carried out. We describe a software program (ChemApplex) based on a calculated parameter (Combined Protein Score) that takes into account (1) peak intensity, (2) the mass accuracy of the match, and (3) ChemScore, a theoretical intensity factor that estimates the probability of observing a particular peptide based on a combination of chemical considerations, in particular the amino acid composition of the peptide and the amino acid sequence of the amino acids that span the cleavage site. When these three factors are taken into account both at the level of individual peptides and at the protein level, protein components in mixtures whose peptides contribute less than 1% of the total intensity can often be correctly identified, as is demonstrated for mixtures of standard proteins. Moreover, it is possible to make robust database identifications that are nearly independent of the number of masses submitted and the mass error threshold used for matching. Protein scoring based on Combined Protein Score is orthogonal to many of the commonly used probability-based scoring schemes, and makes it possible to archive a more complete set of parameters that more thoroughly characterize the validity of the database match, which increases the confidence in the identifications.

Nature of "Tau" immunoreactivity in normal myonuclei and inclusion body myositis.

Sarcoplasmic accumulation of phosphorylated-tau has been widely stated to occur in and contribute to the pathogenesis of muscle disease in inclusion body myositis. Twenty inflammatory myopathy and 10 normal muscle samples along with a range of other tissues were stained with anti-"tau" antibodies (tau-5, pS422, and SMI-31). Myonuclear and sarcoplasmic fractions were prepared using differential solubilization and laser-capture microdissection, and immunoblots were performed using pS422 and SMI-31 antibodies. All three antibodies demonstrated anti-tau immunoreactivity in myonuclei from normal and diseased muscle, but not in nuclei from other tissues. Western blots showed pS422 and SMI-31 immunoreactivity against nuclear proteins outside the region expected for phosphorylated-tau. Antibodies previously reported to indicate abnormal accumulation of phosphorylated-tau in IBM myofibers react to normal myonuclei and recognize proteins other than tau. Normal myonuclei contain neurofilament H or other unidentified 200 kDa proteins with similar phosphorylated motifs accounting for SMI-31 immunoreactivity.

Characterization of human skeletal muscle biopsy samples using shotgun proteomics.

We characterized the human muscle proteome by studying muscle biopsy specimens through four different workflows, using 1 or 2D peptide separation, SDS gels, or differential solubilization. By performing MS/MS analyses of 178 4-h LC separations derived from 31 patients, we identified more than 2000 proteins, and determined how 370 very abundant proteins behave upon differential solubilization. The resulting semiquantitative database should serve as a resource for muscle biochemistry.

Fast-twitch sarcomeric and glycolytic enzyme protein loss in inclusion body myositis.

Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.

Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics.

Protein identification by tandem mass spectrometry is based on the reliable processing of the acquired data. Unfortunately, the generation of a large number of poor quality spectra is commonly observed in LC-MS/MS, and the processing of these mostly noninformative spectra with its associated costs should be avoided. We present a continuous quality score that can be computed very quickly and that can be considered an approximation of the MASCOT score in case of a correct identification. This score can be used to reject low quality spectra prior to database identification, or to draw attention to those spectra that exhibit a (supposedly) high information content, but could not be identified. The proposed quality score can be calibrated automatically on site without the need for a manually generated training set. When this score is turned into a classifier and when features are used that are independent of the instrument, the proposed approach performs equally to previously published classifiers and feature sets and also gives insights into the behavior of the MASCOT score.

Methodology utilizing MS signal intensity and LC retention time for quantitative analysis and precursor ion selection in proteomic LC-MALDI analyses.

This study describes a methodology for performing relative quantitation in large-scale proteomic sample comparisons using an LC-MALDI mass spectrometry analytical platform without the use of isotope tagging reagents. The method utilizes replicate analyses of a sample to create a profile of constituent components that are aligned based on LC elution time and mass. Once components from individual runs have been grouped as common "features", the Student's t test is used to determine which components are systematically different between samples. In this study, five HPLC runs of human plasma were compared to five HPLC runs of human serum. About 3889 components were detected in all 10 runs. Of these, 1831 corresponded to approximately 100 known serum proteins, based on MS/MS analysis of one run each from serum and plasma. As expected, fibrinogen alpha, beta, and gamma chains accounted for many of the most significant differences. Therefore, using MALDI, samples containing thousands of peptides can be compared in a minimal amount of time. Moreover, the results of the comparison can be used to guide further MS/MS mode sample interrogation in a result dependent manner.

Result-driven strategies for protein identification and quantitation--a way to optimize experimental design and derive reliable results.

Uni- or multidimensional microcapillary liquid chromatography (microLC) matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) approaches have gained significant attention for quantifying and identifying proteins in complex biological samples. The off-line coupling of microLC with MS quantitation and MS/MS identification methods makes new result-dependent workflows possible. A relational database is used to store the results from multiple high performance liquid chromatography runs, including information about MALDI plate positions, and both peptide and protein quantitations, and identifications. Unlike electrospray methodology, where all the decisions about which peptide to fragment, must be made during peptide fractionations, in the MALDI experiments the samples are effectively "frozen in time". Therefore, additional MS and MS/MS spectra can be acquired, to promote more accurate quantitation or additional identifications until reliable results are derived that meet experimental design criteria. In the case of what can be designated the expression-dependent workflow, quantitation can be detached from identification and only peak pairs with biological relevant expression changes can be selected for further MS/MS analyses. Alternatively, additional MS/MS data can be acquired to confirm tentative peptide mass fingerprint hits in what is designated a search result-dependent workflow. In the MS data-dependent workflow, the goal is to collect as many meaningful spectra as possible by judiciously adjusting the acquisition parameters based on characteristics of the parent masses. This level of sophistication requires the development of innovative algorithms for these three result-dependent workflows that make MS and MS/MS analysis more efficient and also add confidence to experimental results.

Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins.

Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using crude mouse liver homogenates, four distinct glutathione S-transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The "digital" images generated resemble Western blots without the use of antibodies or signal amplification techniques.

Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.