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Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

ABSTRACT: BACKGROUND: Platelets, the smallest human blood cells component, have a key role in the control of haemostasis and thrombosis but they have also been shown to be implicated in a number of different pathological states because of their involvement also in the process of inflammation end its resolution. Their peculiar anucleated morphology render the proteomics an intriguing approach to understand their biology. Given the high impact of platelet in different diseases we have started a systematic investigation of protein repertoire in controlled platelet preparation. MATERIAL AND METHODS: Platelets have been extracted from blood of healthy donors (n=6) collected by venipuncture in Vacutainer. The quality of the preparation was assessed by observation and enumeration in a Bürker chamber with a conventional tissue culture microscope. To characterize human platelets proteome we analysed the pool of purified platelets combining two proteomic approaches: 2-DE separation combined with Mass Spectrometry and nanoscale ultra performances LC-MS(E) shotgun proteomics experiments. RESULTS: The 2D gel analysis leads an average of 1900 protein spots, after the filtering of "noise" and "false positive" spots, over 500 were selected to be eligible for further analysis given their optimal spot quality value. To perform the analysis by ion accounting shotgun proteomic approach, based on nano ultra performance liquid chromatography (nUPLC) coupled to MS(E) processing of continuum LC-MS data, the same pool of samples was subject to liquid phase tryptic digestion and the peptide obtained used for the experiments. All the data obtained were analysed using ProteinLynx GlobalServer v2.3 (PLGS, Waters). Three analytical replicates run were acquire in high/low energy modes and associated to a human protein database returning the identification of 100 distinct genes. Comparative analysis of the Gene Ontology has been performed to evaluate the differential functional representation of the molecular repertoire investigated with these two orthogonal approaches. DISCUSSION: The overall molecular function classification revealed differences between the two proteomic approaches. In particular, we found significant differences in cytoskeletal proteins (19.65% 2-DE versus 45.60 Shotgun) and receptors (0,92% 2-DE versus 6.90% Shotgun).

SUBMITTER: Finamore F

PROVIDER: S-EPMC2897201 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

Finamore Francesco F Pieroni Luisa L Ronci Maurizio M Marzano Valeria V Mortera Stefano Levi SL Romano Mario M Cortese Claudio C Federici Giorgio G Urbani Andrea A

Blood transfusion = Trasfusione del sangue 20100601

<h4>Background</h4>Platelets, the smallest human blood cells component, have a key role in the control of haemostasis and thrombosis but they have also been shown to be implicated in a number of different pathological states because of their involvement also in the process of inflammation end its resolution. Their peculiar anucleated morphology render the proteomics an intriguing approach to understand their biology. Given the high impact of platelet in different diseases we have started a syste ...[more]

PMID: 20606744

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Project description:Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher's Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples.

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Dataset Information

Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

Publications

Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

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