Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.
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.
Shevchenko
A
A
Protein & Peptide Group, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Chernushevich
I
I
Ens
W
W
Standing
K G
KG
Thomson
B
B
Wilm
M
M
Mann
M
M
D016208:Databases, Factual; D004563:Electrochemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010103:Oxygen Isotopes; D010455:Peptides / Q000032:analysis*; D017421:Sequence Analysis; D014357:Trypsin; D015003:Yeasts / Q000737:chemistry
D007202:Indicators and Reagents; D010103:Oxygen Isotopes; D010455:Peptides; D014357:Trypsin