Delayed Extraction Improves Specificity in Database Searches by Matrix-assisted Laser Desorption/Ionization Peptide Maps

Abstract

Peptide mass maps obtained by matrix-assisted laser desorption ionization (MALDI) are an attractive means to identify proteins by searches in sequence databases. Here we demonstrate that the recently introduced delayed ion-extraction technique, when coupled to reflectron MALDI time-of-flight mass spectrometry, leads to dramatically improved search specificity. Routine resolution in the range of 6,000 to 12,000 allows assignment of monoisotopic masses throughout the peptide mass range. Database searches can be performed with high precision by use of a mass accuracy which is currently better than 30 ppm over a wide mass range and better than 5 ppm for a narrow mass range. This high performance makes it possible to identify proteins with fewer peptide masses than before. Additional low intensity peaks can be assigned after a search because of the improved signal-to-noise ratio of delayed-extraction peptide mass spectra, increasing sequence coverage of matched proteins. The improvements in database search specificity can be used to identify the components of simple protein mixtures. In combination with advanced sample preparation and automation techniques, delayed-extraction MALDI time-of-flight mass spectrometry is now an extremely powerful tool for the database identification of proteins.

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

“…Secondly, most of these relatively low molecular weight proteins (<20 kDa) were predicted to yield only small numbers of peptides from proteolytic digestion with trypsin. For these proteins, identification by PMF often fails since at least 3-5 matching peptides are required for an unambiguous identification (Jensen et al, 1996). This problem has also been observed in a previously published yeast proteome project, where the small and basic proteins such as L41, L40 and L29 were not detected (Link et al, 1999).…”

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure

“…The snR30 and snR42 snoRNP particles were isolated from 100 L of whole-cell yeast extract as described above+ The fractions corresponding to snR30 and snR42 snoRNPs were then phenol/chloroform extracted and the proteins precipitated with acetone+ The resulting proteins were separated on a 13% high-TEMED SDS polyacrylamide gel and stained with colloidal Coomassie blue (Sigma)+ The protein bands were excised from the gel, washed, reduced and alkylated, and finally digested with trypsin as previously described (Shevchenko et al+, 1996)+ After 3 h digestion, 0+3 mL of the supernatant were taken out and used for MALDI peptide mass mapping+ For nanoelectrospray mass spectrometry, the peptides were extracted after digestion over night and desalted into the spraying capillary of the nanoelectrospray source, as described (Shevchenko et al+, 1996;+ Mass spectra were acquired on Bruker REFLEX Matrix Assisted Laser Desorption Ionization time-of-flight mass spectrometer (Bruker-Franzen, Bremen, Germany) equipped with delayed extraction ion source+ The mixture of saturated solution of alpha-cyano hydroxycynnamic acid in acetone and nitrocellulose was used as a thin layer matrix (Vorm et al+, 1994;Jensen et al+, 1996)+ The 0+3 mL of digestion supernatant was deposited into an acidified water droplet on the surface of the matrix (Jensen et al+, 1996)+ Trypsin autolysis products were used for internal calibration that give average accuracy better than 50 ppm+ For identification of proteins using the sequence tag approach (Mann & Wilm, 1994), tandem mass spectra were acquired on an API III triple quadrupole mass spectrometer (Perkin Ellmer-Sciex, Ontario, Canada) equipped with an updated collision cell and a nanoelectrospray source + The peptide ions were sequentially fragmented and from the fragment spectra partial sequence data was obtained for database searching (Mann & Wilm, 1994)+ All proteins were identified using PeptideSearch in nrdb (nonredundant database), which currently holds more than 250,000 entries+…”

Specific antibodies to moesin, a membrane-cytoskeleton linker protein, are frequently detected in patients with acquired aplastic anemia

“…Mass spectrometric identification of an 80-kDa protein derived from the UT-7 cell supernatants was performed as previously described. 18 Briefly, proteins fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis were visualized by Coomassie Brilliant Blue (CBB) staining, and the 80-kDa band was excised from gels, followed by in-gel digestions with trypsin (Promega, Madison, WI) in a buffer containing 50 mM ammonium bicarbonate (pH 8.0) and 2% acetonitrile overnight at 37°C. Molecular mass analyses of the triptic peptides were performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) using an ultraflex TOF/TOF (Bruker Daltonics, Billerica, MA).…”