Protein Differential/Expression Analysis
Relative Qualitative and Quantitative approaches indicating protein levels within the same sample as well as differential levels among samples. A variety of approaches exist within this category -- please read further.
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), as well as “spectral counting” (the number of spectra identified) for a given peptide in different samples. Spectra pertaining to 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 for identification of the peptide sequence; this can pose different issues than MS1 scan label free quantification.
Isobaric tags, namely TMT (tandem mass tags) or iTRAQ (isobaric tags for relative and absolute quantitation) are available. These tags are isobaric in nature, having the same molecular mass, but after isolation and fragmentation (MS/MS) release a “reporter ion”. Isobaric tags afford multiplexing capabilities; currently we support TMT 2- and 6-plex, as well as iTRAQ 4- and 8-plex. These tags work at a “peptide level” and therefore are applicable to almost all experimental scenarios, unlike both label free and SILAC approaches. Please discuss with Ryan Leib details including maximizing proteome depth, co-isolation impurities and kit preferences and purchases.
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 MaxQuant software from the Mann lab. In all instances we rely on our users to troubleshoot and grow cells in 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.