Averagine-scaling analysis and fragment ion mass defect labeling in peptide mass spectrometry

A method termed as the averagine-scaling analysis (ASA) is proposed for predictive design and selection of chemical reagents for modifying peptides, as well as for facile mass spectral analysis of peptide fragment ions with increased mass defects. The ASA method scales mass spectral data using the mass of the hypothetical averagine residue as reference. The scaling analysis is used in conjunction with a strategy of fragment ion mass defect labeling (FIMDL) for effectively using the broad, unoccupied mass zones in the low m/ z region of mass spectra. The FIMDL approach involves the solution modification of peptide termini with chemical reagents of large mass defects and the gas-phase generation of peptide terminal fragment ions that carry the FIMDL groups. The scaling analysis reveals that iodine has the highest FIMDL efficiency among halogens. Iodine-containing reagents, 4-iodophenylisocyanate and 4-iodophenylisothiocyanate, are used to label primary amines on peptides to demonstrate the scaling analysis. The ASA method successfully distinguishes peptide fragment ions with and without an FIMDL group and specifically and efficiently reduces the data complexity of peptide tandem mass spectra. The combination of ASA with FIMDL extends the instrument suitability for the mass defect analysis from mass spectrometers of ultrahigh mass resolution and accuracy to those of medium ones. This combination is expected to have a profound impact on peptide tandem mass spectrometry.