Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry.
Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting 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 and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 degrees C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.
Padliya
Neerav D
ND
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260-3000, USA.
Wood
Troy D
TD
D000595:Amino Acid Sequence; D053000:Analytic Sample Preparation Methods / Q000379:methods*; D003373:Coumaric Acids / Q000378:metabolism; D005841:Gentisates / Q000378:metabolism; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D003373:Coumaric Acids; D005841:Gentisates; D011506:Proteins; C007175:alpha-cyano-4-hydroxycinnamate; D014357:Trypsin; C010925:2,5-dihydroxybenzoic acid