The use of accurate mass tags for high-throughput microbial proteomics.
We describe and review progress towards a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements for microbial systems based upon the use of polypeptide accurate mass tags (AMTs) produced by global protein enzymatic digestions. The two-stage strategy exploits high accuracy mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, from potential mass tags tentatively identified using tandem mass spectrometry (MS/MS), providing the basis for subsequent measurements without the need for routine MS/MS. A high-resolution capillary liquid chromatography separation combined with high sensitivity, and high-resolution accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of more than 10(5) components, sufficient for broad protein identification using AMTs. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, and the capability for stable-isotope labeling methods for precise relative protein abundance measurements. The strategy has been initially evaluated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Additional developments, including the use of multiplexed-MS/MS capabilities and methods for dynamic range expansion of proteome measurements that promise to further extend the quality of proteomics measurements, are also described.
Smith
Richard D
RD
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Anderson
Gordon A
GA
Lipton
Mary S
MS
Masselon
Christophe
C
Pasa-Tolic
Ljiljana
L
Shen
Yufeng
Y
Udseth
Harold R
HR
D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020543:Proteome; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins