Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry.
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 15N-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.
Pasa-Tolić
Ljiljana
L
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Harkewicz
Richard
R
Anderson
Gordon A
GA
Tolić
Nikola
N
Shen
Yufeng
Y
Zhao
Rui
R
Thrall
Brian
B
Masselon
Christophe
C
Smith
Richard D
RD
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D017357:Cyclotrons; D005583:Fourier Analysis; D013058:Mass Spectrometry; D008546:Melanoma, Experimental / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000737:chemistry*; D011492:Protein Hydrolysates / Q000032:analysis; D020543:Proteome / Q000737:chemistry*; D014357:Trypsin
D010455:Peptides; D011492:Protein Hydrolysates; D020543:Proteome; D014357:Trypsin