Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

摘要

This article is the first in a series of reviews intended as a tutorial providing the inexperienced,nas well as the experienced, reader with an overview of principles of peptide and proteinnfragmentation in mass spectrometers for protein identification, surveying of the different typesnof instrument configurations and their combinations for protein identification. The first massnspectrometer was developed in 1899, but it took almost a century for the instrument to becomena routine analytical method in proteomic research when fast atom bombardment ionizationnwas developed, followed shortly by soft desorption/ionization methods, such as MALDI andnelectrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gasnphase under vacuum pressure without destroying them. Thereafter, other soft ionizationntechniques that offered ambient conditions were also introduced, such as atmospheric pressurenMALDI, direct analysisnin real time, atmospheric-pressure solid analysisnprobe and hybridnionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospraynionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spraynionization, desorption electrospray ionization, and electrospray-assisted laser desorptionionization). The five basic types of mass analyzers currently used in proteomic research are thenquadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments,nwhich differ in how they determine the mass-to-charge ratios of the peptides. They have veryndifferent design and performance characteristics. These analyzers can be stand alone or, in somencases, put together in tandem or in conjunction with ion mobility mass spectrometry to takenadvantage of the strengths of each. Several singly or multiply charged fragment ion types, suchnas b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. Innthe bottom-up sequencing approach for protein identification in a shotgun proteomic experiment,nproteolytic digestion of proteins is accomplished by cleavage of the different bonds along thenpeptide backbone and/or side chain through a charge-directed transfer to the vicinity of thencleavage side. These various mass spectrometers and the types of ions produced have becomenimportant analytical tools for studying and analyzing proteins, peptides and amino acids.