An accurate mass tag strategy for quantitative and high-throughput proteome measurements

Abstract

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide "accurate mass tags" (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT-ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 10(5) components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry

“…With the AMT tag approach, it is clear that improvement of the accuracy of mass measurements enables more comprehensive proteome characterization, especially for more complex eukaryotic organisms [21]. However, as shown by our initial studies, there is no "magic" level of mass accuracy that either allows or disallows the use of AMT tags [10]. However, the better the MMA achieved, the more comprehensive the proteome coverage that will be achieved (i.e., the greater the fraction of peptide masses that will be unique within the context of the proteome being studied).…”

“…State-of-the-art technologies for proteomics continually demand higher performance, e.g., high pressure chromatography [8] or use of multiple LC separation dimensions [9]. Our laboratory has focused on an approach using LC separations with highly accurate Fouriertransform ion cyclotron resonance mass spectrometry (FT-ICR) mass spectrometry measure to provide more sensitive and comprehensive proteome analyses based on the concept of accurate mass and time (AMT) tags [10].…”

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

“…Using various statistical and more automated approaches with these tools can improve confidence in identifications, but cannot completely address the issue. In this regard, the use of highly accurate mass measurements provides an additional and high quality "test" for a tentative peptide, and thus increases confidence in identifications [19].…”

Mass-based classification (MBC) of peptides: Highly accurate precursor ion mass values can be used to directly recognize peptide phosphorylation

“…As instruments for determining molecular masses are getting more and more accurate with higher and higher mass resolution, investigation of this structure is becoming an important and fundamental scientific goal. Today, mass accuracies in the range of Ϯ0.2 ppm can routinely be achieved with internal calibration on ion trap Fourier transform ion cyclotron resonance mass spectrometers [1][2][3][4], and it is quite likely that the Ϯ0.1 ppm range of mass accuracies will be accessible in the foreseeable future even with external calibration.The structure of molecular masses can be distinguished into a coarse, fine, and hyperfine structure, similar to the spectroscopic structure of molecules. Introducing a phosphorylation, for example, leads to a shift of the distribution to a higher mass deficit (to smaller masses), but does not create a separation of the distribution of phosphorylated peptides into the "forbidden zone" of the distribution of unmodified peptides (Figure 1b).…”

“…As instruments for determining molecular masses are getting more and more accurate with higher and higher mass resolution, investigation of this structure is becoming an important and fundamental scientific goal. Today, mass accuracies in the range of Ϯ0.2 ppm can routinely be achieved with internal calibration on ion trap Fourier transform ion cyclotron resonance mass spectrometers [1][2][3][4], and it is quite likely that the Ϯ0.1 ppm range of mass accuracies will be accessible in the foreseeable future even with external calibration.…”