Biomedical Engineering Reference
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all peptide ion currents relative to the total ion current can be
used as a measure of protein amounts in absolute units. Nonethe-
less, regardless of its use as an absolute quantification strategy,
the usefulness of label-free LC-MS for relative quantification of
proteins is now well accepted by the proteomics community and
has also been used to quantify phosphorylation sites on proteins
A major problem for the implementation of label-free quanti-
tative LC-MS based on ion currents is that of choosing an appro-
priate strategy for data analysis. Computationally, it is more dif-
ficult to compare LC-MS runs than to evaluate the signals of
groups of ions in the same MS trace, as in label-based experi-
ments. This is because even with the most accurate nanoflow LC
systems, changes in retention times across samples are unavoid-
able, especially when nanoflow columns are loaded with concen-
trated samples containing large amounts of peptides (in these
cases of nearly overloading, interactions of peptides may account
for differences in retention times in consecutive LC-MS runs).
We have occasionally observed that up to 4 min shift in retention
time in consecutive LC-MS runs (100-min gradient) when using
a modern ultrahigh-pressure liquid chromatography system. This
lack of absolute reproducibility is not a problem for label-based
techniques because comparison of ion intensities is performed
within the same MS data files, but it can affect the results of label-
free LC-MS experiments.
Different strategies are currently available to the researcher
in order to overcome difficulties in the analysis of label-free data;
these include chromatogram alignment, generation of XIC of pre-
viously identified peptides, or using an MRM approach.
Comparison of aligned ion chromatograms
. A common
strategy for comparing LC-MS runs is to align and compare chro-
matograms resulting from the analysis of the samples to be evalu-
are compared using statistical tests. The identities of differentially
expressed peptide ions are then obtained by including their masses
and retention times in an inclusion list for targeted LC-MS/MS
analysis. Procedures for alignment of chromatograms are not
straightforward because differences in RT across samples are not
always linear; RT offsets may be a few minutes in one direction at
the beginning of the gradient, while they could be on the other
direction at the end of it. This was the same problem that the
2D gel community faced when this technique was in its infancy,
and the same solutions that were found to align 2D gels may also
be used to align chromatograms. In fact, chromatogram align-
ment may be less problematic than aligning 2D gels because only
RT may vary across samples (
m
/
z
values can be calculated with
high degree of accuracy by modern mass spectrometers), whereas,
because of variability of gel electrophoresis, spots in 2D gels vary
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