PMID- 17696519
OWN - NLM
STAT- MEDLINE
DCOM- 20071108
LR  - 20070907
IS  - 1535-3893 (Print)
IS  - 1535-3893 (Linking)
VI  - 6
IP  - 9
DP  - 2007 Sep
TI  - Contribution of protein fractionation to depth of analysis of the serum and 
      plasma proteomes.
PG  - 3558-65
AB  - In-depth analysis of the serum and plasma proteomes by mass spectrometry is 
      challenged by the vast dynamic range of protein abundance and substantial 
      complexity. There is merit in reducing complexity through fractionation to 
      facilitate mass spectrometry analysis of low-abundance proteins. However, 
      fractionation reduces throughput and has the potential of diluting individual 
      proteins or inducing their loss. Here, we have investigated the contribution of 
      extensive fractionation of intact proteins to depth of analysis. Pooled serum 
      depleted of abundant proteins was fractionated by an orthogonal two-dimensional 
      system consisting of anion-exchange and reversed-phase chromatography. The 
      resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun 
      LC-MS/MS, and another was further resolved into protein bands in a third 
      dimension using SDS-PAGE. Individual gel bands were excised and subjected to in 
      situ digestion and mass spectrometry. We demonstrate that increased fractionation 
      results in increased depth of analysis based on total number of proteins 
      identified in serum and based on representation in individual fractions of 
      specific proteins identified in gel bands following a third-dimension SDS gel 
      analysis. An intact protein analysis system (IPAS) based on a two-dimensional 
      plasma fractionation schema was implemented that resulted in identification of 
      1662 proteins with high confidence with representation of protein isoforms that 
      differed in their chromatographic mobility. Further increase in depth of analysis 
      was accomplished by repeat analysis of aliquots from the same set of 
      two-dimensional fractions resulting in overall identification of 2254 proteins. 
      We conclude that substantial depth of analysis of proteins from milliliter 
      quantities of serum or plasma and detection of isoforms are achieved with 
      depletion of abundant proteins followed by two-dimensional protein fractionation 
      and MS analysis of individual fractions.
FAU - Faca, Vitor
AU  - Faca V
AD  - Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, 
      Washington 98109, USA. vfaca@fhcrc.org
FAU - Pitteri, Sharon J
AU  - Pitteri SJ
FAU - Newcomb, Lisa
AU  - Newcomb L
FAU - Glukhova, Veronika
AU  - Glukhova V
FAU - Phanstiel, Doug
AU  - Phanstiel D
FAU - Krasnoselsky, Alexei
AU  - Krasnoselsky A
FAU - Zhang, Qing
AU  - Zhang Q
FAU - Struthers, Jason
AU  - Struthers J
FAU - Wang, Hong
AU  - Wang H
FAU - Eng, Jimmy
AU  - Eng J
FAU - Fitzgibbon, Matt
AU  - Fitzgibbon M
FAU - McIntosh, Martin
AU  - McIntosh M
FAU - Hanash, Samir
AU  - Hanash S
LA  - eng
PT  - Journal Article
DEP - 20070816
PL  - United States
TA  - J Proteome Res
JT  - Journal of proteome research
JID - 101128775
RN  - 0 (Anions)
RN  - 0 (Blood Proteins)
RN  - 0 (Protein Isoforms)
RN  - 0 (Proteins)
RN  - 0 (Proteome)
SB  - IM
MH  - Anions
MH  - Blood Proteins/*chemistry
MH  - Chromatography, Ion Exchange/methods
MH  - Chromatography, Liquid/methods
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Humans
MH  - Lung Neoplasms/metabolism
MH  - Mass Spectrometry/methods
MH  - Protein Isoforms
MH  - Proteins/*chemistry
MH  - Proteome
MH  - Proteomics/*methods
MH  - Serum/metabolism
EDAT- 2007/08/19 09:00
MHDA- 2007/11/09 09:00
CRDT- 2007/08/19 09:00
PHST- 2007/08/19 09:00 [pubmed]
PHST- 2007/11/09 09:00 [medline]
PHST- 2007/08/19 09:00 [entrez]
AID - 10.1021/pr070233q [doi]
PST - ppublish
SO  - J Proteome Res. 2007 Sep;6(9):3558-65. doi: 10.1021/pr070233q. Epub 2007 Aug 16.