Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins.
Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.
Rose
Keith
K
GeneProt, 2 rue Pré-de-la-Fontaine, CP125, CH-1217, Meyrin 2, Switzerland. keith.rose@geneprot.com
Bougueleret
Lydie
L
Baussant
Thierry
T
Böhm
Günter
G
Botti
Paolo
P
Colinge
Jacques
J
Cusin
Isabelle
I
Gaertner
Hubert
H
Gleizes
Anne
A
Heller
Manfred
M
Jimenez
Silvia
S
Johnson
Andrew
A
Kussmann
Martin
M
Menin
Laure
L
Menzel
Christoph
C
Ranno
Frederic
F
Rodriguez-Tomé
Patricia
P
Rogers
John
J
Saudrais
Cedric
C
Villain
Matteo
M
Wetmore
Diana
D
Bairoch
Amos
A
Hochstrasser
Denis
D
D000595:Amino Acid Sequence; D001774:Blood Chemical Analysis / Q000379:methods*; D001798:Blood Proteins / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019295:Computational Biology; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010949:Plasma / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001798:Blood Proteins; D007074:Immunoglobulin G; D010455:Peptides; D020543:Proteome; D014357:Trypsin