iTRAQ-labeling of in-gel digested proteins for relative quantification.

In addition to standard MS-based protein identification, quantification of proteins by mass spectrometry (MS) is rapidly gaining acceptance in proteomic studies. MS-based quantification involves either the incorporation of stable isotopes or can be performed label-free. Recently, more attention has been devoted to label-free quantification; however, this approach has not been fully established among the proteomic community yet. More common is still the introduction of stable isotopes, which can be done by metabolic (e.g., SILAC) or by chemical (e.g., ICAT, iTRAQ, etc.) labeling. Here, we present an overall quantification strategy for chemical labeling of in-gel digested proteins using iTRAQ reagents. This includes (1) protein separation by gel electrophoresis, (2) excision of protein bands, (3) in-gel digestion and extraction of peptides, (4) labeling of peptides, (5) pooling the samples to be compared, (6) LC-MS/MS of labeled peptides, and (7) database search. The presented workflow is well suited for protein samples of moderate complexity (i.e., protein samples of 300-400 components), and it is exemplified by using different amounts of 25S [U4/U6.U5] tri-snRNPs.