scholarly journals Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis

2020 ◽  
Vol 21 (11) ◽  
pp. 3971
Author(s):  
Martin Ondrej ◽  
Pavel Rehulka ◽  
Helena Rehulkova ◽  
Rudolf Kupcik ◽  
Ales Tichy
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Reversed Phase ◽  
Chromatographic Technique ◽  
Phosphopeptide Enrichment ◽  
Ms Analysis ◽  
Input Material ◽  
Fractionation Method ◽  
Microcolumn Separation ◽  
Gas Tight

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC–MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC–MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

scholarly journals Proteomics Analyses of the Opportunistic PathogenBurkholderia vietnamiensisUsing Protein Fractionations and Mass Spectrometry

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Samanthi Wickramasekara ◽  
Julie Neilson ◽  
Naren Patel ◽  
Linda Breci ◽  
Amy Hilderbrand ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Virulence Factors ◽  
Protein Identification ◽  
Reversed Phase ◽  
Tandem Mass ◽  
Burkholderia Vietnamiensis ◽  
Ms Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Identification Technique

The main objectives of this work were to obtain a more extensive coverage of theBurkholderia vietnamiensisproteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome ofB. vietnamiensiswas precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the proteins prior to reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The second method used MudPIT analysis (Multi dimensional Protein Identification Technique), where proteins are digested and separated using cation exchange and reversed phase separations before the MS/MS analysis (LC/LC-MS/MS). Overall, gel-based LC-MS/MS analysis resulted in more protein identifications than the MudPIT analysis. Combination of the results lead to identification of more than 1200 proteins, approximately 16% of the proteins coded from the annotated genome ofBurkholderiaspecies. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases.

scholarly journals Improvement of Capture Compound Mass Spectrometry Technology (CCMS) for the Profiling of Human Kinases by Combination with 2D LC-MS/MS

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Jenny J. Fischer ◽  
Olivia Graebner ◽  
Mathias Dreger ◽  
Mirko Glinski ◽  
Sabine Baumgart ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Small Molecule ◽  
Protein Interactions ◽  
Protein Identification ◽  
Spectrometric Analysis ◽  
Proteomic Profiling ◽  
Protein Conjugates ◽  
Input Material ◽  
Mass Spectrometry Technology

An increasingly popular and promising field in functional proteomics is the isolation of proteome subsets based on small molecule-protein interactions. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate captured protein conjugates from complex biological samples for direct protein identification by liquid chromatography/mass spectrometry (nLC-MS/MS). In this study we used staurosporine as a selectivity group for analysis in HepG2 cells derived from human liver. In the present study, we combined the functional isolation of kinases with different separation workflows of automated split-free nanoflow liquid chromatography prior to mass spectrometric analysis. Two different CCMS setups, CCMS technology combined with 1D LC-MS and 2D LC-MS, were compared regarding the total number of kinase identifications. By extending the chromatographic separation of the tryptic digested captured proteins from 1D LC linear gradients to 2D LC we were able to identify 97 kinases. This result is similar to the 1D LC setup we previously reported but this time 4 times less input material was needed. This makes CCMS of kinases an even more powerful tool for the proteomic profiling of this important protein family.

Mass Spectrometry Techniques: Principles and Practices for Quantitative Proteomics

2020 ◽  
Vol 21 ◽  
Author(s):  
Rocco J. Rotello ◽  
Timothy D. Veenstra
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Protein Identification ◽  
Dynamic Nature ◽  
Complete Coverage ◽  
The Past ◽  
Proteome Level ◽  
A Cell ◽  
Quantitative Changes ◽  
Made In

: In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and metabolites present within a cell. While genomics has led to a dramatic increase in our understanding of such things as disease morphology and how organisms respond to medications, it is critical to obtain information at the proteome level since proteins carry out most of the functions within the cell. The primary tool for obtaining proteome-wide information on proteins within the cell is mass spectrometry (MS). While it has historically been associated with the protein identification, developments over the past couple of decades have made MS a robust technology for protein quantitation as well. Identifying quantitative changes in proteomes is complicated by its dynamic nature and the inability of any technique to guarantee complete coverage of every protein within a proteome sample. Fortunately, the combined development of sample preparation and MS methods have made it capable to quantitatively compare many thousands of proteins obtained from cells and organisms.

scholarly journals The Analysis of Bayer Liquor by SPME-GC-MS of Derivatized Organic Poisons

Proceedings ◽  
2021 ◽  
Vol 57 (1) ◽  
pp. 101
Author(s):  
Virgil Badescu ◽  
Raluca Senin
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Gas Chromatography Mass Spectrometry ◽  
Bayer Liquor ◽  
Chromatography Mass Spectrometry ◽  
Ms Analysis ◽  
Silylating Agent

The aim of this article was the gas chromatography–mass spectrometry (GC-MS) analysis oforganic matter from a residual liquor sample (S.C. Alum S.A., Tulcea), extracted by the solid-phasemicroextraction method (SPMA) and derivatized with N-(tert-butyldimethylsilyl)-Nmethyltrifluoroacetamide(MTBSTFA) as the silylating agent. [...]

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