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J Biomol Tech. 2007 February; 18(1): 89–90.
PMCID: PMC2292016

EP6 Quantitative Proteomics

Abstract

There are numerous approaches to study the proteome in a quantitative manner. All rely heavily on optimized sample preparation and appropriate statistical analysis of resulting datasets. This session will cover the following aspects of quantitative proteomics approaches:

  1. Quantitative profiling of the membrane proteome requires special considerations not addressed in typical mass-spectrometry analyses. Optimized sample preparation and separation strategies will be discussed in the context of enriched membrane fractions and a quantitative proteomics platform using stable isotopes.
  2. In shotgun proteomics, a complex protein mixture is first digested to peptides, which are then analyzed by a combination of nanoflow chromatography and tandem mass spectrometry. The effects of subtle changes in sample preparation and chromatographic conditions in the characterization of complex mixtures will be presented. A discovery-based mass spectrometry approach using a bench-top LTQ linear ion trap and in-house written software for label-free differential protein profiling will be presented. This approach is quite comprehensive and is compatible with even the most inexpensive mass spectrometers. For proteins not detected routinely using our discovery-based approaches, we have applied selected reaction monitoring using a TSQ Quantum Ultra. This approach has been used to identify and quantify proteins at the low ng/mL level in plasma without any prior fractionation. A software pipeline has been developed to go from hypothesized proteins of interest derived from the literature to predicted hSRM transitions, collision offsets, and predicted chromatographic retention times. The combination of both discovery- and hypothesis-driven proteomics using nanoflow separations and tandem mass spectrometry provides us with unparalleled sensitivity and dynamic range in characterizing complex mixtures.
  3. Spectrum counting is an appealing and relatively straightforward approach for quantitative proteomics. Since the spectrum count of a protein in a proteomic analysis is the total number of peptides, not just unique peptides detected and identified for a given protein, searching criteria and false-positive minimization is important. There are several different versions of spectral counting currently in use, but each approach has shared core characteristics. An additional important consideration for quantitative proteomic analysis is the use of replicates for statistical analysis and determining the proper statistical test to use based on the overall structure of the datasets. This presentation will describe the foundation of spectral counting and the modifications to this approach used by different researchers. In addition, selected examples of the biological implementation of these approaches will be described.

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities