Application of MS Methodologies for the Analysis of PTM Status

Knowledge of the precursor mass and the diagnostic ion facilitates analytical approaches, both discovery and targeted. The specific pair of m/z values associated with a precursor and product ion (including the PTM diagnostic ion) is termed a “transition" which provides the basis for “reaction monitoring" Selected reaction monitoring (SRM)/multiple reaction monitoring (MRM) are based on iterative precursor isolation and scanning of MS2 transition ions, typically using triple quadrupole MS, where the Q1 and Q3 mass analyzers are used as static mass filters for the selected m/z values of precursor-product transition ions. CID occurs in Q2. MRM-initiated detection and sequencing (MIDAS) is a technique for PTM analysis [47] with application to protein acetylation based on precursor-126.1 transition [48]. This approach is more specific and 10-fold more sensitive than precursor ion scanning for 126.1 ion, where precursor masses are scanned in Q1, with Q3 static for the product ion mass [49, 50]. MIDAS employs a hybrid triple quadrupole/linear ion trap to enable full product ion scan information - triggered by the detection of PTM diagnostic. The MIDAS approach requires a priori knowledge of the protein sequence for targeted analysis of the predicted, theoretical precursor-PTM diagnostic ion transitions.

An alternative approach, collision-induced release of acetyl diagnostic (CIRAD) MS method, utilizes the acetylation diagnostic ion for discovery of acetylation in a knowledge-independent manner to assign acetylated precursor m/z [51]. This is achieved by the use of broadband CID, rapid cycling between low and elevated collision energies to generate intact and fragment high- resolution mass spectra (<2 ppm accuracy) from all precursors simultaneously. The presence of the 126.091 m/z fragment ion is monitored and aligned to the precursor m/z using an accurate mass-retention time information-type approach. Processing of the LC-MS raw data with a specific “dissect” extraction algorithm links precursors and product ions (pseudo tandem MS information for database searching) by retention time, traced across the LC elution peak using both the high- and low-energy data. The CIRAD approach complements and informs other MS analysis methods by providing precursor m/z, precursor-product transitions, and retention time information independent of primary sequence or information on PTM status.

< Prev   CONTENTS   Source   Next >