A strategy to improve peptide mass fingerprinting matches through the optimization of matrix-assisted laser desorption/ionization matrix selection and formulation

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides that result from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly‐charged ions, along with its relative insensitivity to salts and buffers, matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) is the MS method of choice for PMF. The qualitative features of a MALDI‐MS mass spectrum can be selectively tuned by varying the matrix and the solvent system used to prepare the matrix. The selective tuning of MALDI‐MS mass spectra in order to optimize PMF results is addressed in this paper. Carbonic anhydrase, hemoglobin α‐ and β‐chain, and myoglobin were digested with trypsin, and the digest was analyzed with MALDI‐MS. 2,5‐Dihydroxybenzoic acid (2,5‐DHB) and α‐cyano‐4‐hydroxycinnamic acid were prepared, using five different solvent systems: (A) 99% acetone; (B) 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA); (C) 75% ACN, 0.1% TFA; (D) formic acid:H2O: 2‐propanol (1:3:2); and (E) H2O:MeOH (2:1). Each protein was found to have a different optimum solvent system for PMF. Generally, better PMF results were obtained with 2,5‐DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest were convolved.