De novo sequencing of tryptic peptides derived from Deinococcus radiodurans ribosomal proteins using 157 nm photodissociation MALDI TOF/TOF mass spectrometry.
Vacuum ultraviolet photodissociation of peptide ions in a matrix assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer is used to characterize peptide mixtures derived from Deinococcus radiodurans ribosomal proteins. Tryptic peptides from 52 proteins were separated by reverse-phase liquid chromatography and spotted onto a MALDI plate. From 192 sample spots, 492 peptide ions were isolated, fragmented by both photodissociation and postsource decay (PSD), and then de novo sequenced. Three-hundred seventy-two peptides yielded sequences with 5 or more amino acids. Homology searches of these sequences against the whole bacterial proteome identified 49 ribosomal proteins, 45 of which matched with two or more peptides. Peptide de novo sequencing identified slightly more proteins than conventional database searches using Mascot and was particularly advantageous in identifying unexpected peptide modifications. In the present analysis, 52 peptide modifications were identified by de novo sequencing, most of which were not recognized by database searches.
Zhang
Liangyi
L
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Reilly
James P
JP
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D056148:Chromatography, Reverse-Phase; D030562:Databases, Protein; D034301:Deinococcus / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry; D040901:Proteomics / Q000379:methods; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D010446:Peptide Fragments; D012269:Ribosomal Proteins; D014357:Trypsin