A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high‐resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1‐DE) followed by na...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2005-08, Vol.5 (13), p.3329-3342
Main Authors: Tang, Hsin-Yao, Ali-Khan, Nadeem, Echan, Lynn A., Levenkova, Natasha, Rux, John J., Speicher, David W.
Format: Article
Language:English
Subjects:
Biomarkers - chemistry
Blood Proteins - chemistry
Blood Proteins - isolation & purification
Chromatography, Liquid
Humans
Hydrogen-Ion Concentration
Isoelectric Focusing
Liquid chromatography-tandem mass spectrometry
Mass Spectrometry
Peptides - chemistry
Peptides - isolation & purification
Plasma proteome
Protein Array Analysis
Protein arrays
Proteins - chemistry
Proteins - isolation & purification
Proteomics - methods
Serum proteome
Statistics as Topic
Time Factors
Trypsin - pharmacology
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Summary:A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high‐resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1‐DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2‐D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO‐approved criteria for high‐confidence protein assignments. Most importantly, a substantial number of low‐abundance proteins (low ng/mL – pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 µL (45 µg) of plasma and 2.4 µL (204 µg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.200401275