Multidimensional Protein Identification Technology Approach Research Articles

Patterns of Proteins Removed with High-Flux Membranes on High-Volume Hemodiafiltration Detected with a MultiDimensional LC-MS/MS Strategy

Background: Aim of this prospective crossover study was to identify the nature of the middle-molecular weight solutes removed during high-volume post-dilution HDF. Methods: The efficiency in removing small molecules, protein-bound and middle-molecular proteins was evaluated in 16 chronic dialysis patients on post-dilution HDF with two high-flux dialyzer membranes (Amembris and Polyamix). Multidimensional Protein Identification Technology (MudPIT) was employed to identify middle-molecular weight solutes in spent dialysate. Results: Efficiency of post-dilution HDF in removing solutes of different MW was high with both membranes, but higher with Amembris than with Polyamix. With MudPIT analysis, 277 proteins were identified in the dialysate fluids. Although the protein-removal pattern was similar among patients and tested membranes, the total and protein-specific peptide spectral count (mass spectrometric quantitation criteria) of most proteins were higher using the Amembris membrane. Conclusions: The MudPIT approach showed to be a powerful tool to identify a broad molecular weight spectrum of proteins removed with post-dilution HDF. Short Summary: Aim of this prospective crossover study was to analyze the hydraulic properties of two high-flux dialyzer membranes (Amembris and Polyamix) during high-volume, post-dilution HDF and to evaluate the influence of these properties on the removal of proteins and peptides using an in-depth analysis of the spent dialysate. For this analysis, a liquid chromatography tandem mass spectrometry approach called MudPIT (Multidimensional Protein Identification Technology) was used to identify the middle molecular weight solutes present in the spent dialysate of patients. The capability of post-dilution HDF in removing solutes of different MW was very high with both dialyzers, but higher with the Amembris membrane. The proteomic MudPIT approach showed to be a powerful tool to identify a wide molecular spectrum of proteins removed from blood during post-dilution HDF. These results may contribute to address research toward a better knowledge of uremic toxins and the balance between the intended and unintended removal of undesired and beneficial proteins next to identification of new target proteins as potential candidates for uremic toxicity. i 2014 S. Karger AG, Basel

Proteome profile of a pandemic Vibrio parahaemolyticus SC192 strain in the planktonic and biofilm condition

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors’ knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.

Clinical Proteomics

Clinical proteomics involves the analysis of protein expression of disease proteomes, with the aim of solving a specific clinical problem. Discoveries made from proteomicbased studies contribute to the growing need for innovative medical diagnostics for disease detection. Taking into consideration the global health burden of cardiac disease, clinical proteomics is a valuable tool to improve risk stratification associated with this disease. In cardiovascular medicine, the identification of novel proteins, or biomarkers, that are differentially expressed in cardiac disease proteomes may enable early detection of the disease state, thereby preventing progression to disease end points. This review outlines various proteomic platforms and their technical advancements and relates these to the cardiovascular sciences. The entire protein complement of the cell, or proteome, is dynamic and changes in response to the disease state. 1 Proteomic-based experiments can be used to characterize such alterations in protein expression during disease progression. 2 With combined improvements in mass spectrometry (MS) technology as well as innovative molecular biology screening tools, there has been widespread growth in the characterization of cardiac disease proteomes. In fact, proteomic technology has been an important tool in the analysis of heart failure (HF), 3,4 cardiac hypertrophy, 5,6 and dilated cardiomyopathy.7 Several of the overarching aims of such studies include providing greater understanding of general biological mechanisms as well as identifying unique proteins that are clinically useful in the detection of cardiac disease in the early stages or potentially used as novel therapeutic targets. Clinical proteomics continues to benefit from advancements in technologies that allow for fast and consistent identification of proteins with corresponding increases in the dynamic range of proteins detectable in the disease proteome. MS-related technologies have improved in their ability to detect low-abundance proteins as well as membrane proteins and have benefited from sample preprocessing strategies that decrease the complexity of large-scale analyses. With the emergence of different methodologies for biomarker identification, the following 3 criteria must be taken into careful consideration to determine whether the protein is, in fact,

A Novel Multidimensional Protein Identification Technology Approach Combining Protein Size Exclusion Prefractionation, Peptide Zwitterion−Ion Hydrophilic Interaction Chromatography, and Nano-Ultraperformance RP Chromatography/nESI-MS2 for the in-Depth Analysis of the Serum Proteome and Phosphoproteome: Application to Clinical Sera Derived from Humans with Benign Prostate Hyperplasia

The current proof-of-principle study was aimed toward development of a novel multidimensional protein identification technology (MudPIT) approach for the in-depth proteome analysis of human serum derived from patients with benign prostate hyperplasia (BPH) using rational chromatographic design principles. This study constituted an extension of our published work relating to the identification and relative quantification of potential clinical biomarkers in BPH and prostate cancer (PCa) tissue specimens. The proposed MudPIT approach encompassed the use of three distinct yet complementary liquid chromatographic chemistries. High-pressure size-exclusion chromatography (SEC) was used for the prefractionation of serum proteins followed by their dialysis exchange and solution phase trypsin proteolysis. The tryptic peptides were then subjected to offline zwitterion-ion hydrophilic interaction chromatography (ZIC-HILIC) fractionation followed by their online analysis with reversed-phase nano-ultraperformance chromatography (RP-nUPLC) hyphenated to nanoelectrospray ionization-tandem mass spectrometry using an ion trap mass analyzer. For the spectral processing, the sequential use of the SpectrumMill, Scaffold, and InsPecT software tools was applied for the tryptic peptide product ion MS(2) spectral processing, false discovery rate (FDR) assessment, validation, and protein identification. This milestone serum analysis study allowed the confident identification of over 1955 proteins (p ≤ 0.05; FDR ≤ 5%) with a broad spectrum of biological and physicochemical properties including secreted, tissue-specific proteins spanning approximately 12 orders of magnitude as they occur in their native abundance levels in the serum matrix. Also encompassed in this proteome was the confident identification of 375 phosphoproteins (p ≤ 0.05; FDR ≤ 5%) with potential importance to cancer biology. To demonstrate the performance characteristics of this novel MudPIT approach, a comparison was made with the proteomes resulting from the immunodepletion of the high abundant albumin and IgG proteins with offline first dimensional tryptic peptide separation with both ZIC-HILIC and strong cation exchange (SCX) chromatography and their subsequent online RP-nUPLC-nESI-MS(2) analysis.

Automated 2D Peptide Separation on a 1D Nano-LC-MS System

Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two-dimensional precolumn in a vented column setup. When compared to the traditional MudPIT approach, this nanoflow variation demonstrated better first-phase separation leading to more proteins being characterized while using rather simple instrumentation and a protocol that requires less time and very little technical expertise to perform.

Multidimensional protein identification technology for clinical proteomic analysis

Proteomics technologies demonstrate enormous data-gathering capabilities to discover disease-specific biomarkers in serum, plasma, urine, tissue and other biological samples. The traditional way to achieve this consists of protein separation performed using two-dimensional polyacrylamide gel electrophoresis (2DE). However, the 2DE approach has its drawbacks with respect to automation, sensitivity, and throughput. Considerable efforts have been devoted to the development of non-gel-based proteome separation technologies able to resolve complex protein and peptide mixtures prior to mass spectrometric (MS) analysis. This review discusses some of the most recent advances in multidimensional peptide separation techniques compatible with MS analysis, including gel electrophoresis, isoelectric focusing, capillary electrophoresis and liquid chromatography techniques. Based on future perspectives and our experiences, special attention is given to the application of two-dimensional chromatographic separation coupled to MS, the so-called multidimensional protein identification technology approach.

Sample preparation and in-solution protease digestion of proteins for chromatography-based proteomic analysis.

The adoption of chromatography-based proteomics approaches has resulted in the development of new methods for optimal use of these technologies. One such technology, named multidimensional protein identification technology (MudPIT), directly couples liquid chromatography with tandem mass spectrometry. In the MudPIT approach, digested protein samples are directly loaded onto a microcapillary column packed sequentially with reversed-phase and strong cation exchange resins. Once digested protein samples are loaded onto the column, the column is placed in-line between a high-performance liquid chromatography system and an electrospray ionization tandem mass spectrometer (ESI-MS/MS). The digested protein samples for MudPIT analysis must be directly compatible with ESI-MS/MS, so the sample preparation and digestion protocols must be optimized for this purpose. The primary objective of all of the protocols in this unit is to yield a final digested protein mixture of less than 300 microl that can then be directly loaded onto a MudPIT column.

Association of Catsper1 or -2 with Cav3.3 Leads to Suppression of T-type Calcium Channel Activity

Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca(v)3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca(v)3.3-evoked T-type calcium current without altering other biophysical properties of Ca(v)3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca(v)3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca(v)3.3 channel, which might be important in the regulation of sperm function.

Protein sequence tags: A novel solution for comparative proteomics

Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins. This procedure addresses a similar number of proteins, compared to the multidimensional protein identification technology approach, but offers additionally a quantitative analysis with its recently developed quantitative PST version.

A proteomic view ofBifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gut where they exert several health-promoting effects. The present paper reports the use of a strong cation exchange-reversed-phase-tandem mass spectrometry strategy to catalogue the most abundantly expressed proteins of a probiotic Bifidobacterium infantis strain. A global view of the B. infantis proteome was obtained. The bimodal representation of the proteins identified by mass spectrometry provides the first theoretical two-dimensional map of protein distribution for this organism. Among the 136 proteins identified by multidimensional protein identification technology (MudPIT) analysis, 118 showed the highest similarity with the translated sequences of B. longum genome, two proteins were similar to other Bifidobacterium species and the remaining 16 were similar to different genera. Specific biological activities have been assigned to 115 identified proteins, whereas 21 have been referred to the group of hypothetical proteins. The MudPIT approach allowed us to identify high mass and basic isoelectric point proteins that are generally challenging to visualize using the traditional two-dimensional electrophoresis technique. Redundancy in peptide and protein identification using the double chromatography technique was also evaluated.