Fractionation of Isotopically Labeled Peptides in Quantitative Proteomics

Abstract

The goal of quantitative proteomics is to examine the expression levels of all of the proteins in a biological system and recognize those that change as a function of some stimulus. Quantification is now frequently based on derivatization of peptides with isotopically distinguishable labeling agents. This study examines the extent to which isotopic forms of peptides having the same amino acid sequence are resolved by reversed-phase chromatography and assesses the degree to which resolution of these isotopically different forms of a peptide impact quantification. Three derivatizing agents were examined, the do and d3 forms of N-acetoxysuccinimide, the do and d4 forms of succinic anhydride, and the do and d8 forms of the commercial ICAT reagent Peptide mixtures from control and experimental samples were derivatized individually, mixed, subjected to reversed-phase chromatography, and analyzed by ESI-MS. When partial resolution of the isotopic forms of a peptide occurs, the largest error in assessing the true isotope ratio in a sample occurs when sampling at the extremes of a peak. Early in the elution of a peak, the sample will be enriched in the deuterated species, whereas the opposite is true at the tailing edge of a peak. Acetylated peptides showed the lowest degree of separation. Resolution of the deuterated and nondeuterated forms in this case was 0.023. This amounts to slightly over a 1-s difference in their peak maxima and can cause a typical error of +/- 6% at the leading and tailing edges of a peak. In contrast, resolution of the deuterated and nondeuterated forms of the ICAT reagent were calculated to be 0.45. This means that in a peak of 1-min width (W1/2), the peak maxima will vary by approximately 30 s, and measurement errors of -83 and +500% can occur at the leading and tailing edges of a peak. It is concluded that resolution of isotopic forms of a peptide can cause substantial quantification errors in quantitative proteomics.

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

“…Such fractionation depends on mass difference between unlabeled and 2 H-labeled molecules, as was previously observed for 2 H-coded peptides when 2 H 8 -ICAT reagent was used in comparative proteomics (35). In theory, the chromatographic fractionation of deuterated peptides could complicate the analysis of isotopic distribution (36).…”

Protein profiling of sickle cell versus control RBC core membrane skeletons by ICAT technology and tandem mass spectrometry

“…Molloy et al [7] treated E. coli with Triclosan to inhibit the expression of Fab I, which is involved in fatty acid synthesis, and determined a variation on average of 22.6% in their treated/control ratios. The use of the second generation acid cleavable 12 C/ 13 C cysteine labeling cICAT reagents addresses the slight delay of the heavy and light cICAT tagged peptides eluting from a C18 reverse phase column by removing biotin prior to LC MS/MS [6,[17][18][19]. The cICAT technology was designed to address the documented weaknesses of 2D SDS PAGE for protein separation [20][21][22][23] such as precipitation of hydrophobic proteins (GRAVY>0.3) and heavily glycosylated proteins, inability to resolve proteins with very small (<10 kDa) or very large (>150 kDa) molecular weights; inability to resolve proteins with very high or very low isoelectric point; and a limited dynamic range.…”

What Can Proteomics Tell Us About Platelets?

“…(1) Currently multiplexing is limited to only 3 samples and, as for all nonisobaric labels, 2-plexing will double and 3-plexing will triple the complexity of the sample. (2) Incorporation of deuterium causes retention time shifts between light/medium/ heavy forms of the peptide during reversed phase chromatography and thus can complicate quantification by liquid chromatography-MS. 65 Hebert et al 66,67 recently introduced a novel strategy for neutron-encoded labeling, which will increase the multiplexing capabilities of the aforementioned labeling strategies, but requires mass spectrometers with a resolution power of ≈500 000 and above, currently supported by only few instruments in conjunction with reasonable sensitivity and scanning speed. (6) with subsequent multiplexing and analysis of pooled samples can be applied.…”