Protocols to Assure Reproducible Quantitative and Qualitative Analysis of Tryptic Digests of Complex Protein Mixtures for Global Proteomics Experiments

Library Number:
720000894EN
Part Number:
720000894EN
Author(s):
Craig A. Dorschel, Marc V. Gorenstein, Guo-Zhong Li, Jeffrey C. Silva, Scott Geromanos, and Timothy Riley
Source:
Waters ASMS 2004 poster
Content Type:
Posters
Content Subtype:
ASMS
To obtain meaningful results in qualitative and quantitative proteomics, such as observation of different levels of expression of one or more proteins in a set of samples and identification of the proteins, it is necessary that great care be taken in preparation of samples and in obtaining exact mass LC/MS data. Digestions with proteases such as trypsin should be carried to completion, so that the ratio of concentrations of fragment peptides will accurately reflect the ratio of concentration of the original protein in two samples. Accurate measurement of peptide masses allows more stringent searching of protein databases resulting in faster and more accurate qualitative identification of proteins. Ionization of peptides should be consistent from run to run for valid quantitative comparisons. Maximizing the reproducibility of chromatographic retention times simplifies the overall task of correlating the massive data sets obtained in global proteomic experiments. We have developed protocols and software tools to enable us to reproducibly digest complex protein mixtures, routinely obtain precise mass measurements on peptides (typically less than/equal to 5 ppm error), minimize run-to-run retention time drift at the capillary separation scale, and demonstrate the quality and reproducibility of our results. We illustrate this with digestion and analysis of replicate samples of rat serum using the Waters Protein Expression System.

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