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

Abstract

A primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. 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 show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis. (J Am Soc Mass Spectrom 2002, 13, 954 -963)

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

“…To quench any remaining ion populations in this region of the selection quadrupole, the potential at the middle segments was increased to 20 V during ion trapping in the ICR cell. This selective ion ejection approach enables the DREAMS capability that has been previously shown to enhance the dynamic range of FTICR measurements [34,35,49].…”

“…Removal of these major species and the subsequent accumulation of lower abundance species for extended periods results in an increase in dynamic range. DREAMS expanded the dynamic range of FTICR to Ͼ10 5 , resulting in a 40% increase over conventional LC/FTICR detection in the number of identified putative peptides from a yeast tryptic digest [34,35]. Shi et al [36] showed that higher mass resolution can be achieved by lowering trapping potentials to produce a very shallow trapping well and the minimization of space charge (e.g., Coulomb repulsion) effects.…”

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

“…In addition, the use of accurately predicted LC elution times [16] could be an effective way to increase confidence and reduce the false positive rate by further limiting the number of candidates for peptides eluting at a given time. Finally, we strongly believe that with the use of DREAMS [21,22] and related data-dependent methods for smarter selection of peptides for dissociation [1], the benefits of multiplexed MS/MS would be entirely realized. Specifically, this would allow the biologically relevant (i.e.…”

Combining low and high mass ion accumulation for enhancing shotgun proteome analysis by accurate mass measurement

“…Peptide identification is usually achieved by tandem mass spectrometry [4 -6], or more recently, by accurate mass measurement using Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry [2,3,[7][8][9][10][11][12][13]. In the accurate mass measurement approach, proteins are identified by matching the measured peptide mass values with those predicted from an in silico digest of all proteins from the subject genome [2,3,[7][8][9][10]. Since the identification relies only on mass values, the throughput of this approach is considerably higher because more than 100 peptide masses per mass spectrum can be measured and identified, while tandem mass spectrometry approach requires a one-by-one analysis of each peptide.…”

“…In addition, the accurate mass measurement approach is less biased toward more abundant peptides compared with tandem mass spectrometry identification, in which the most abundant peptides are generally selected for analysis by the software that controls the online HPLC MS/MS measurement. The accurate mass measurement approach provides a high throughput alternative, and reliable quantification can be made with stable isotope labeling [3,7,8,10]. The peptide identification specificity is directly related to the mass accuracy level that serves as the primary search constraint.…”