Review: The Use of Accurate Mass Tags for High-Throughput Microbial Proteomics

Abstract

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.

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

“…The structure and data input of the accurate mass and time tag database previously developed using LC-FTICR instrumentation has been previously described [22,26]. The AMT tag database operationally utilizes a Microsoft SQL server and contains information describing the peptide, ORF (open reading frame) reference, mass spectral, separation, and other tracking and sample specific data.…”

“…In the AMT tag approach [21] to proteomics involves the combined use of accurate mass measurement and LC elution time information to increase peptide identification confidence [22]. The AMT tag approach is well suited to take advantage of the improving accuracies provided by TOF instruments.…”

Correlation of mRNA Expression and Protein Abundance Affected by Multiple Sequence Features Related to Translational Efficiency in Desulfovibrio vulgaris: A Quantitative Analysis

“…H IGH-THROUGHPUT postgenomic technologies such as microarray and proteomics analyses have provided powerful methodologies to study patterns of gene expression and regulation at the genome scale (Horak and Snyder 2002;Smith et al 2002). Given the fact that no single approach can fully unravel the fundamental biology that is typically quite complex, an integrative approach of multiple levels of information is necessary and valuable to fully elucidate the complex biological system being studied.…”

Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry

“…Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15 N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion.…”

“…We have recently described a global proteomics strategy that provides improvements in sensitivity, dynamic range, comprehensiveness, and throughput based upon the use of polypeptide "accurate mass tags" (AMTs), i.e., peptides (or modified peptides) with the molecular masses measured with high enough mass measurement accuracy (MMA) such that their masses are effectively unique among all of the possible peptides predicted from an annotated genome [13][14][15][16]. The two-stage strategy initially described exploits high resolution capillary LC separations combined with Fourier transform ion cyclotron resonance (FTICR) instrumentation [17], to first validate polypeptide AMTs using accurate mass measurements combined with MS/MS measurements.…”