Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

Abstract

Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/ MS spectra were clustered, combined, and searched against the IPI protein databank using PepMiner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.

Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly‐available database

“…Barnea et al [30] expanded on their original submission as Lab 1 (Tables 1 and 2) with an analysis of several protein fractionation and several MS/MS methods on PPP reference specimen B2-serum. Albumin and IgG were depleted with are Bio-Rad mini-kit based on Affi-Gel Blue and Affi-Gel protein A, respectively.…”

“…To test the feasibility of this approach, we searched all ORFs using peak list data from six PPP laboratories (17,30,37,41,52,55). NCBI human genome sequence build 33 was translated in all three reading frames and both strands; all non-redundant ORFs were assembled into chromosome specific sequence collections.…”

Human body fluid proteome analysis

“…An important observation was that each of the methods revealed a set of unique proteins. Therefore, for comprehensive identification of plasma/serum proteins, several different pre-fractionation methods should be used in parallel [44]. Liquidphase IEF (ampholyte-free) appeared to be very effective for prefractionation of peptides.…”

Proteomic analysis optimization: Selective protein sample on-column retention in reverse-phase liquid chromatography☆

“…This was not surprising because of the broad dynamic range of proteins in biological tissues and an automatic (data dependent) peak selection and exclusion used in the MS/MS analysis. However, eventually a plateau in the number of protein identifications would be expected for multiple replicate protein fractionation experiments carried out prior to the mass spectral analysis [26].…”