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Rapid Digestion and Reproducible LC-MS quantification of Cytochrome C: A Potential Biomarker for Apoptosis

Rapid Digestion and Reproducible LC-MS Quantification of Cyt...

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2018年中国质谱学术大会（CMSC2018）

作者： Paula Orens Mary Lame Erin Chambers Waters Corporation

Cytochrome C, is a mitochondrial protein(～13 kDa) which plays important roles in oxidative phosphorylation and apoptosis, or programmed cell death. Elevated plasma concentrations(～2 μg/mL) of circulating cytochrome C have been reported in patients with conditions associated with mitochondrial damage1. Use of LC-MS analysis for protein quantification has become more popular in the past few years due to the many benefits it offers(e.g., multiplexing, improved specificity, broader linear dynamic range, and faster method development times). However, for protein quantification by LC-MS, these protein digestion workflows are complex and time consuming, with enzymatic digestions often taking upwards of 24 hours to achieve sensitive and accurate quantification from complex biological matrices. Thus, We developed a rapid digestion and reproducible LC-MS method for the accurate quantification of cytochrome C in plasma. 35 μL plasma sample were digested for 10 minutes using proteinWorks e Xpress Direct Digest Kit. Post digestion purification of signature peptides was completed using the proteinWorks μElution SPE Clean-up Kit(15 minute SPE). Four unique tryptic peptide were identified: TGPNLHGLFGR, MIFAGIK, EDLIAYLK, and GITWGEETLMEYLENPKK. Chromatographic separation of cytochrome C tryptic peptides was achieved using a CORTECS UPLC C18+, 1.6 μm, 2.1 mm x 50 mm Column(5 minute method), which provides excellent peak shape, narrow peak widths(quantification for Four unique signature tryptic peptide was 2 μg/ml. For all four peptides, at all QC levels, samples demonstrated excellent accuracy and precision with an average %CV of 2.5%. This work demonstrates proof of concept for the rapid, accurate and precise quantification of the protein biomarker, cytochrome C. The combination of the

关键词： cytochrome C protein quantification proteinWorks μElution SPE CORTECS UPLC C18+

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SILVER:A Software Tool for Stable Isotope Labeling LC-MS data analysis in Quantitative Proteomics with Visual and Effective Function

SILVER:A Software Tool for Stable Isotope Labeling LC-MS dat...

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第五届全国生物信息学与系统生物学学术大会暨国际生物信息学前沿研讨会高通量时代的生物信息学与系统生物学

作者： Cheng Chang Jiyang Zhang Hanchang Sun Ping Xu Hongwei Xie Yunping Zhu State Key Laboratory of Proteomics Beijing Proteomc Research CenterBeijing Institute of Radiation Medicine Department of Automatic Control College of Mechatronies and AutomationNational University of Defense TechnologyChangshaHunan 410073China

Background:With the advance of experimental technologies,large-scale protein quantification is widely applied to sample analysis in *** stable isotope labeling methods have been developed for the comparison of different samples on the mass spectrometry(MS) *** software tools have been developed for single or multiple labeling ***,there are few software tools available that can support most of the widely used labeling techniques,and has easily accessible graph user interface (GUI) and fairish computation speed. Methods:Here,we present an effective tool for protein quantification based on the stable isotope labeling MS data,which introduces a compact Index MS raw data file(IdxRaw) to accelerate the quantitative information extraction,a least square fitting with nonlinear optimization algorithm for the universal quantitative calculation on single MS spectrum from different isotopic labeling techniques.S/N ratio and R-square of the linear fitting were calculated as filter criteria during the construction of extracted ion chromatogram(XIC).Then, linear regression normalization and Dixon’s Q-test are applied for combining the peptide ratio in reduplicate runs.A protein ratio was defined as a weighted average of unique peptide ratios. Results:In this tool,batch processing for the large scale data analysis and GUI for the manual inspection and validation are *** user can easily trace the details of protein quantification,including the extracted ion chromatogram(XIC) for each peptide,the paired isotopic peaks in the MS spectrum and several parameters for the result *** merits of SILVER were verified by applying it to datasets with different isotopic labeling. Conclusions:SILVER shows high accuracy and relative robustness on both the peptide level and the protein ***’s more,it also includes a user-friendly visualization platform, which permits users to validate the results.

关键词： Bioinformatics Quantitative Proteomics protein quantification Stable isotope labeling

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