Snapp: Simplified nanoproteomics platform for reproducible global proteomic analysis of nanogram protein quantities

Abstract

Global proteomic analysesofcomplex protein samplesin nanogram quantities requireafastidious approach to achieve in-depth protein coverage and quantitative reproducibility. Biological samples are often severely mass limited and can preclude the application of more robust bulk sample processing workflows. In this study, we present a system that minimizes sample handling by using onlineimmobilizedtrypsindigestionandsolidphaseextractiontocreateasimple,sensitive,robust, and reproducible platform for the analysis of nanogram-size proteomic samples. To demonstrate the effectiveness of our simplified nanoproteomics platform, we used the system to analyze preimplantation blastocysts collected onday4 of pregnancy by flushing the uterine horns with saline. For each of our three sample groups, blastocysts were pooled from three mice resulting in 22, 22, and 25 blastocysts, respectively. The resulting proteomic data provide novel insight into mouse blastocyst protein expressionon day 4 of normal pregnancy because we characterized 348 proteins that were identified in at least two sample groups, including 59 enzymes and blastocyst specific proteins (eg, zona pellucida proteins). This technology represents an important advance in which future studies could perform global proteomic analyses of blastocysts obtained from an individual mouse, thereby enabling researcherstoinvestigate interindividual variationaswellas increase the statistical power without increasing animal numbers. This approach is also easily adapt able to other mass-limited sample types.