Solid phase isobaric mass tag reagent for simultaneous protein identification and assay.

The solid phase isobaric mass tagging (SPIMT) approach is presented for simultaneous protein quantitation and identification. The novelty of the SPIMT strategy relies on a CID-based differentiation of regioisomeric species for quantitation of tagged proteolytic peptides. SPIMTs are unlabeled mass-tagging reagents, which consist of a reporter group, a mass balance group, and a spacer with a amine-specific reactive group, able to be linked to any N-terminal peptide. Therefore SPIMT-linked peptides from a two-plex set appear as a single unresolved precursor ion in MS, whereas the reporter groups lead to quantitation signals of m/z 168.2 and 182.2 Da upon tandem mass spectrometry (MS/MS) analysis with matrix-assisted laser desorption time-of-flight/time-of-flight (MALDI TOF/TOF). This strategy allows ease protein identification by direct submission of MS and MS/MS data to the MASCOT database. SPIMT approach showed an excellent quantitation linearity, detecting any relative concentration differences of peptides in two solutions over a 5-fold concentration range without losing sequencing information. Therefore, SPIMTs are an attractive, simple, and low cost alternative for two-plex quantitation of proteins and offer possibilities of tuning the two-plex signal mass window by replacing the spacer.