A brief introduction is presented to the basic principles and application of a quadrupole-time-of-flight (TOF) tandem mass spectrometer. The main features of reflecting TOF instruments with orthogonal injection of ions are discussed. Their operation and performance are compared with those of triple quadrupoles with electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometers. Examples and recommendations are provided for all major operational modes: mass spectrometry (MS) and tandem MS (MS/MS), precursor ion scans and studies of non-covalent complexes. Basic algorithms for liquid chromatography/MS/MS automation are discussed and illustrated by two applications.

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 hybrid quadrupole time-of-flight mass spectrometer featured with ion trapping capabilities was employed for quantitative profiling of total extracts of endogenous phospholipids. Simultaneous acquisition of precursor ion spectra of multiple fragment ions allowed detection of major classes of phospholipids in a single experiment. Relative changes in their concentration were monitored using a mixture of isotopically labeled endogenous lipids as a comprehensive internal standard. Precursor ion scanning spectra were acquired simultaneously for acyl anions of major fatty acids in negative ion mode and identified the fatty acid moieties and their relative position at the glycerol backbone in individual lipid species. Taken together, a combination of multiple precursor ion scans allowed quantitative monitoring of major perturbation in phospholipid composition and elucidating of molecular heterogeneity of individual lipid species.

Collisional cooling of ions in the rf-only multipole guides has become a method of choice for coupling electrospray sources to various mass analyzers. Normally parameters of such ion guides (length, pressure) provide enough thermalization and focusing for ions in a wide mass range. Noncovalent complexes, however, have more compact conformations than denatured biomolecules of similar mass and, therefore may not be transmitted efficiently through standard ion guides, as demonstrated by theoretical analysis, simulations, and experiments. Several methods of improving collisional cooling for large compact ions have been developed on a quadrupole time-of-flight instrument, which include operating the ion guides at higher pressure and trapping ions to increase the cooling time. Improved transmission of heavy ions obtained with those methods is studied in experiments with proteasome 20S, an oligomeric protein noncovalent complex with molecular weight around 692,000, and a few other compounds.

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