A molecular scanner to automate proteomic research and to display proteome images.
Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annoated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html + ++), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.
Binz
P A
PA
Swiss Institute of Bioinformatics, University Medical Center, Geneva, Switzerland. Pierre-Alain.Binz@dim.hcuge.ch
Müller
M
M
Walther
D
D
Bienvenut
W V
WV
Gras
R
R
Hoogland
C
C
Bouchet
G
G
Gasteiger
E
E
Fabbretti
R
R
Gay
S
S
Palagi
P
P
Wilkins
M R
MR
Rouge
V
V
Tonella
L
L
Paesano
S
S
Rossellat
G
G
Karmime
A
A
Bairoch
A
A
Sanchez
J C
JC
Appel
R D
RD
Hochstrasser
D F
DF
D001331:Automation; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis / Q000737:chemistry
D001798:Blood Proteins; D014360:Trypsin Inhibitor, Kunitz Soybean