Protein Differential/Expression Analysis

Relative Qualitative and Quantitative approaches indicate protein levels within the same sample as well as differential levels among samples. A variety of approaches exist within this category:

Modern proteomics dictates that protein identification is only part of the puzzle, and increasingly it is necessary to understand the expression profiles of protein(s) within the overall context of the cell. Multiple solutions exist, some more accurate, robust, and applicable than others. Our lab has experience with several methodologies, including:

Label-free quantification is available using both precursor ion intensity (MS1 scan), and “spectral counting” (the number of spectra identified) for a given peptide in different samples. Spectra of a particular protein are summed and normalized to quantify the protein(s). Both strategies are available to our users.   Spectral counting relies on the MS2 scan, which is used to identify the peptide sequence; this can pose different issues than MS1 scan label-free quantification.

SILAC (stable isotope labeling with amino acids in cell culture) is a robust analytical methodology for quantitative proteomics in which cells are cultured in media containing heavy (13C-incorporated lysine or arginine) or light media (normal media). Typically, after 6-7 cell cycles, the amino acid is fully incorporated and the light and heavy forms can be pooled, digested, and analyzed. Because the protease of choice is trypsin or LysC, we can be assured that all peptides will contain a “labeled” amino acid. Data analysis relies on Spectronaut software. In all instances, we rely on our users to troubleshoot and grow cells under the necessary conditions. SILAC is limited to specific cell lines and cannot be applied to tissue samples.  The Pandey lab has many great tips that can be found here: http://silac.org/recipes.