Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10(3). High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.
We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) CE-MS/MS procedure which employs a high sensitivity porous ESI sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.
MicroRNAs (miRNAs) regulate specific immune mechanisms but their genome-wide regulation of T-lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T-lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T-lymphocytes) using locked nucleic acid inhibitors. Inhibition of these 2 highly upregulated miRNAs in CD4+ T cells were shown to increase proliferation by removing suppression of 4 target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T-lymphocyte immunity underlining the value of mapping global gene, protein and miRNA expression.
The most common cause of kidney transplant failure is the poorly characterized histopathologic entity interstitial fibrosis and tubular atrophy (IFTA). There are no known unifying mechanisms, no effective therapy, and no proven preventive strategies. Possible mechanisms include chronic immune rejection, inflammation, drug toxicity, and chronic kidney injury from secondary factors. To gain further mechanistic insight, we conducted a large-scale proteogenomic study of kidney transplant biopsies with IFTA of varying severity. We acquired proteomic data using tandem mass spectrometry with subsequent quantification, analysis of differential protein expression, validation, and functional annotations to known molecular networks. We performed genome-wide expression profiling in parallel. More than 1400 proteins with unique expression profiles traced the progression from normal transplant biopsies to biopsies with mild to moderate and severe disease. Multiple sets of proteins were mapped to different functional pathways, many increasing with histologic severity, including immune responses, inflammatory cell activation, and apoptosis consistent with the chronic rejection hypothesis. Two examples include the extensive population of the alternative rather than the classical complement pathway, previously not appreciated for IFTA, and a comprehensive control network for the actin cytoskeleton and cell signaling of the acute-phase response. In summary, this proteomic effort using kidney tissue contributes mechanistic insight into several biologic processes associated with IFTA.
Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.