MALDI-quadrupole time-of-flight mass spectrometry was applied to identify proteins from organisms whose genomes are still unknown. The identification was carried out by successively searching a sequence database-first with a peptide mass fingerprint, then with a packet of noninterpreted MS/MS spectra, and finally with peptide sequences obtained by automated interpretation of the MS/MS spectra. A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions. This approach was tested in a small-scale proteomic project involving the identification of 15 bands of gel-separated proteins from the methylotrophic yeast Pichia pastoris, whose genome has not yet been sequenced and which is only distantly related to other fungi.
The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560-to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R 2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano-to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier. Molecular & Cellular Proteomics 3:908 -919, 2004.The application of MS to biomolecular analysis has revolutionized protein research within the past decade (1). This can be mostly attributed to the development of ionization techniques that are compatible with biomolecules, i.e. MALDI (2, 3) and ESI (4), as well as improved instrumentation. However, although modern mass spectrometers provide high mass accuracy and sensitivity, the protein complexity and concentration range usually found in biological samples still present a challenge. The problem has been traditionally attacked by separation of complex protein mixtures by two-dimensional gel electrophoresis, with subsequent protein in-gel digestion, followed by ESI or MALDI MS. This remains one of the most popular sample preparation procedures, especially suitable for protein identification and quantitation. However, the method is best suited for higher abundance proteins with masses greater than 12-14 kDa, and some categories of molecules, such as membrane proteins (1) or species with extremes in isoelectric points, are handled poorly. There are also difficulties in adapting the method to high-throughput applications.Alternative analytical approaches are based on pre-fractionation of protein mixtures or cell lysates before the final MS steps of analysis (5-9). This often involves proteolytic digestion, followed by one-or multi-dimensional chromatographic separation of the resulting peptides, with subsequent detection by MS/MS. Such a method may yield considerable simplification of the problem, because the fractions from on-or off-line HPLC separations have reduced complexity compared with the original sample. Indeed, the combination of HPLC-ESI (MS or MS/MS) has proved to be a "work horse" for large-scale high-throug...
Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.
A matrix‐assisted laser desorption/ionization (MALDI) source has been coupled to a tandem quadrupole/time‐of‐flight (QqTOF) mass spectrometer by means of a collisional damping interface. Mass resolving power of about 10,000 (FWHM) and accuracy in the range of 10 ppm are observed in both single‐MS mode and MS/MS mode. Sub‐femtomole sensitivity is obtained in single‐MS mode, and a few femtomoles in MS/MS mode. Both peptide mass mapping and collision‐induced dissociation (CID) analysis of tryptic peptides can be performed from the same MALDI target. Rapid spectral acquisition (a few seconds per spectrum) can be achieved in both modes, so high throughput protein identification is possible. Some information about fragmentation patterns was obtained from a study of the CID spectra of singly charged peptides from a tryptic digest of E. coli citrate synthase. Reasonably successful automatic sequence prediction (>90%) is possible from the CID spectra of singly charged peptides using the SCIEX Predict Sequence routine. Ion production at pressures near 1 Torr (rather than in vacuum) is found to give reduced metastable fragmentation, particularly for higher mass molecular ions. © 2000 John Wiley & Sons, Ltd.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.