Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer

Abstract

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.

Formation of (bn−1 + H2O) ions by collisional activation of maldi-formed peptide [M + H]+ ions in a QqTOF mass spectrometer

“…Mass spectra of whole digest mixtures were recorded with our Manitoba/SCIEX prototype tandem quadrupole/time-of-flight mass spectrometer°(QqTOF)°(PE/SCIEX,°Concord,°ON,°Canada)° [38], which was coupled to a MALDI ion source for these experiments° [39,°40],°as°shown°schematically°in°Figure S1 (Supplemental). The matrix was prepared from a 100 mg/mL solution of 2,5-dihydroxybenzoic acid (DHB) in acetone/water (1:1 vol/vol).…”

Oxidation–reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts

“…Proteins were subjected to in-gel trypsin digestion [32]. The excised gel spots were then destained using 100 mL of destaining solution (1:1 = 30 mM potassium ferricyanide:100 mM sodium thiosulfate, v/v) with agitation for 5 min.…”

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

“…Others have already exploited this feature of peak doublets to do peptide de novo sequencing by applying a 50:50 16 O/ 18 O labeling of the C-termini that results in a mass difference of 2 Da [25,26]. By recognizing y-ions this way, we were for instance able to deduce easily the sequence SVYADQ/K from the MS/MS spectrum acquired on the ESI-QTOF and the ESI-IT of the plasma peptide shown in Figure 3 with a monoisotopic mass of 1708.5 ( Figure 5).…”