Multidimensional protein identification technology (MudPIT) is
another related approach to automated and integrated whole
proteome expression analysis exploiting mass spectrometry and
its ability to identify peptides. In this approach, whole proteomes
or fractions are digested and separated using a dual-phase liquid
chromatography separation process followed by in-line analysis
by electrospray ionization mass spectrometry.16,17 This approach
differs from other approaches in that no sampling of peptides is
performed. MudPIT relies on high-resolution multidimensional
chromatography to resolve all the peptide components of a
complex mixture. This technique has the advantage of complete
representation of proteins in the original mixture, but at the cost
of high redundancy, which results in high sample complexity and
limits throughput.
In addition, labeling techniques to allow relative abundances
of proteins in different samples to be determined by mass
spectrometry are also needed. A number of such labeling
techniques, notably ICAT, have also been published recently.12,18-20
These techniques are all based on standard procedures for
quantification by mass spectrometry in which an analyte is
quantified by comparison with an introduced isotopomer of the
analyte that acts as an internal standard. Typically, the internal
standard is differentiated from the analyte by incorporation of
deuterium, but 13C and 15N are also used. It is assumed in these
techniques that the relative signal intensities of analyte and
standard are directly proportional to their relative concentrations,
and since the quantity of the standard is known, this means that
the quantity of the analyte may be determined from the ratios of
their peak intensities. These standard methods of quantification
have been adapted for the purpose of protein expression analysis
by the introduction of “heavy” and “light” isotope tags that are
used to label peptides from corresponding proteins in pairs of
samples under comparison. The isotope tagging procedures
produce pairs of labeled peptide isotopomers that are mass-
differentiated and can act as mutual internal standards. The ICAT
procedure, in particular, combines a method of sampling a
complex protein mixture with a method of determining relative
abundances by using pairs of isotope-differentiated cysteine-
reactive affinity tags and may be regarded as the most advanced
alternative to 2D-PAGE for whole proteome analysis to date. The
MudPIT procedure is also compatible with the isotope labeling
techniques that have been developed recently.21
isotopically labeled nutrients that will be incorporated into proteins
into the growth medium of a living organism. Clearly, this
approach is limited to the analysis of organisms whose growth
media can be controlled. Moreover, in this approach, the precise
amount of incorporation of heavy and light isotopes cannot really
be predicted. In vivo approaches are typified by the complex
spectra of the mass modified peptides. While these patterns can
be useful for the identification of some peptides, the complexities
and limitations of this approach make it laborious and, thus,
unattractive. In addition, the organism must be grown on minimal
media, which has metabolic implications for the experimental
organism; i.e., the protein expression patterns on minimal media
will be a specific response to the medium, and these patterns will
bias the response behavior of the model organism. Consequently,
minimal media cannot permit the full range of protein expression
to be explored. In contrast, in vitro labeling allows labeling of
virtually any protein sample, it allows control over the degree of
isotope labeling of each peptide, and the conditions under which
the sample proteins are produced do not affect the labeling. In
vitro labeling procedures are, thus, more appealing than in vivo
procedures as long as robust labeling protocols can be developed.
Despite some successes with the isotope labeling techniques
discussed above, all of the published approaches that depend
on deuterium labeling suffer from a number of problems. The
most significant is that, although the mass modification that results
from isotope labeling is small, there is still a detectable shift in
the mobility of deuterium differentiated peptides in size/ mass-
dependent separation procedures, such as reversed-phase high
performance liquid chromatography (HPLC).12,22 Typically, the
heavy peptide migrates more rapidly than the light peptide, often
eluting as a separate fraction.12 This means that in order to
accurately determine the quantities of each heavy/ light peptide
pair, it is necessary to allow both peptides to completely elute to
allow integration of the ion current for each peptide. As a
consequence, the determination of the peptide identity by se-
quencing using MS/ MS techniques cannot easily be reconciled
with the need for accurate abundance measurements. Moreover,
since each peptide pair does not coelute, the isotope-tagged
peptides do not act as true standards for each other, reducing
confidence in the accuracy of relative quantification. In particular,
it is possible that one peptide of a pair, but not the other, may
coelute with another peptide that suppresses its ionization.
Another problem for quantitative analysis of peptides labeled
with conventional isotope labels using LC/ MS arises from the
different charge states of the labeled peptides that are produced
by electrospray ionization. This means that the mass difference
between corresponding peptides labeled with conventional isotope
tags varies with the charge state of the peptide. Similarly, the
number of tags incorporated into a peptide will alter the mass
difference between each corresponding peptide from a paired
sample.
The currently published “heavy/ light” isotope labeling tech-
niques fall into two general categories: in vivo19,20 and in vitro12,18
labeling. The former approach requires the introduction of
(14) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M.
M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2 0 0 2 , 20,
301-305.
(15) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2 0 0 1 , 19, 379-382.
(16) Washburn, M. P.; Yates, J. R. Curr. Opin. Microbiol. 2 0 0 0 , 3, 292-297.
(17) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2 0 0 1 , 19, 242-
247.
(18) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.;
Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass
Spectrom. 2 0 0 1 , 15, 1214-1221.
Improved labels can solve some of the problems with the above
techniques, for example, the novel 13C reagent for ICAT described
by ABI avoids the retention time shift.
(19) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad.
Sci. U.S.A. 1 9 9 9 , 96, 6591-6596.
This paper describes a novel class of reagents termed tandem
mass spectrometry tags (TMTs), where the term “tandem” refers
(20) Pasa-Tolic, L.; Jensen, P. K.; Anderson, G. A.; Lipton, M. S.; Peden, K. K.;
Martinovic, S.; Tolic, N.; Brice, J. E.; Smith, R. D. J. Am. Chem. Soc. 1 9 9 9 ,
121, 7949-7950.
(21) Washburn, M. P.; Ulaszek, R.; Deciu, C.; Schieltz, D. M.; Yates, J. R., III.
(22) Griffin, T. J.; Han, D. K.; Gygi, S. P.; Rist, B.; Lee, H.; Aebersold, R.; Parker,
Anal. Chem. 2 0 0 2 , 74, 1650-1657.
K. C. J. Am. Soc. Mass Spectrom. 2 0 0 1 , 12, 1238-1246.
1896 Analytical Chemistry, Vol. 75, No. 8, April 15, 2003