42 BIOCHEMISTRY FUNDAMENTALS
to known proteins in mixtures, but these are unlikely to provide unequivocal
protein identifi cation. One method used to identify individual proteins has
been to excise a spot containing one protein from the 2 - D gel and then digest
the protein with a proteolytic enzyme to form peptide fragments which are
then analyzed by mass spectrometry (MS). Peptide mass profi les obtained by
MS analysis of given proteins can yield unique profi les leading to protein
identifi cation when compared directly to known protein MS peptide mass
profi le databases. One may also use Edman degradations — the same chemical
method described above for successively releasing individual N - terminal resi-
dues of a protein — followed by MS of the degradation products to obtain
peptide mass profi les of the protein. These methods are slow if one wishes to
identify the hundreds of different proteins that may be separated on the 2 - D
gel.
The proteomics mass spectrometric and other analytical techniques must
be capable of high - throughput sensitive screening of proteins separated on 2 -
D gels so that only those proteins that cannot be identifi ed unequivocally or
appear to be novel need further characterization by protein sequencing. High -
pressure liquid chromatographic (HPLC) analyses assist here as some proteins
have unique amino acid compositions that can be used for their identifi cation
when compared to databases of protein amino acid compositions. Gel electro-
phoresis, MS, and HPLC techniques are continually being refi ned and extended
for more effi cient proteome analyses. One fi nal requirement for proteomic
technology is that the generated data must be stored in databases that can be
interrogated effectively in the laboratory and made available to other scien-
tists worldwide through the internet or other mechanisms. Lists of known 2 - D
PAGE (two - dimensional polyacrylamide gel electrophoresis) protein data-
bases are maintained at http://www.expasy.ch/ch2d/2d-index.html. The Swiss
Proteomics Society maintains a page with links to publications and informa-
tion in the proteomics area at http://www.swissproteomicsociety.org/links.
html.
The proteomics research of a number of scientists was described in a C & E
News report of the 2001 Pittcon meeting.^9 One group, that of Catherine Fense-
lau at the University of Maryland, studied a new method for proteolytic stable
isotope labeling to provide quantitative and concurrent comparisons between
individual proteins from two entire proteome pools.^10 Two serotypes of an
adenovirus were selected for study. Two O^18 atoms were incorporated into the
carboxyl termini of tryptic peptides (peptides digested using trypsin) during
the proteolytic cleavage of all proteins in the fi rst pool. Proteins in the second
pool were also cleaved using trypsin digestion with the carboxyl termini of the
resulting peptides containing two O^16 atoms (no labeling). The two peptide
mixtures (^18 O mixture plus^16 O mixture) were pooled for fractionation and
separation by HPLC, and the masses and isotope ratios of each peptide pair
(differing by 4 Da) were measured by high - resolution mass spectrometry.
Short sequences and/or accurate mass measurements combined with pro-
teomics software tools allowed the peptides to be related to the precursor