Peptide fractionation in proteomics approaches

Manadas, Bruno; Mendes, Vera M.; English, James; Dünn, Michael J.

doi:10.1586/epr.10.46

Cited by 59 publications

(43 citation statements)

References 36 publications

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“…The reduced number of identifications observed in comparison to DT phase samples is likely due to 1) AQ phase sample complexity, as indicated by protein banding pattern in Figure 2, and 2) the protein yield of 1.36 mg which was considerably larger than that of the DT phase at 0.45 mg. It’s possible that a 2-Dimensional fractionation of AQ peptides, prior to MS, would improve identifications [31]. 2D-LC-MS/MS would increase the orthogonality of peptide elution, simplifying the mixture and thus increasing the number of protein identifications [32]. These results further highlight the need to pre-fractionate complex protein samples prior to in-depth LC-MS/MS analysis for successful biomarker identification.…”

Section: Discussionmentioning

confidence: 99%

Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

et al. 2012

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Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

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Section: Discussionmentioning

confidence: 99%

Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

et al. 2012

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“…Of the many types of fractionation techniques [82-84], strong cation exchange (SCX), strong anion exchange…”

Section: Ms-based Methodologies For the Study The Aml Proteomementioning

confidence: 99%

Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients

Aasebø¹,

Forthun²,

Berven³

et al. 2015

CPB

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The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging.

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“… “The introduction of multidimensional peptide resolving techniques is of unquestionable value for the characterization of complex proteomes” [35] …”

Section: Peptide Fractionation Proceduresmentioning

confidence: 99%

“…The introduction of ion-pair reagents increased the retention of charged analytes and improved peak shapes. Trifluoroacetic acid (TFA) and formic acid (FA) have been extensively used as ion-pairing reagents [35]. …”

Section: Peptide Fractionation Proceduresmentioning

confidence: 99%

Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

Abdallah

Dumas‐Gaudot

Renaut³

et al. 2012

International Journal of Plant Genomics

168

175

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Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.

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Peptide fractionation in proteomics approaches

Cited by 59 publications

References 36 publications

Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients

Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

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