Protein Protocols Protein Protocols

538 Stone and Williams
(while maintaining a constant gradient time program) eventually, the linear flow velocity
on the column will be reduced to such an extent that optimal resolution will be lost (3–5).
At this point, the column diameter needs to be decreased so that a more optimal linear
flow velocity can be maintained at a lower flowrate. In general, the sensitivity of detection
is increased as the wavelength is decreased with the practical limit in 0.05% TFA being
about 210 nm. Finally, an important determinant of sensitivity (that is often overlooked) is
the path length of the flow cell. For instance, an HP1090 equipped with a 0.6-cm path length
cell provides (at the same flowrate) a threefold increase in sensitivity over that afforded
by a Michrom UMA System equipped with a 0.2-cm path length cell.
2. If the transit time (i.e., peak delay) between the detector and the fraction collector is too
long, closely eluting peaks will be pooled together. The reason is that if a second peak is
detected by the Isco Model 2150 Peak Separator while it is “counting down” the peak
delay for the first peak, the two peaks will be pooled together. Although our experience is
this phenomenon seldom occurs with a peak delay of 6 s (at a flowrate of 75 µL/min),
which corresponds to a “dead volume” of about 7.5 µL, it often occurs if the peak delay
exceeds about 15 s. To our knowledge, no commercial peak detector is currently available
that can simultaneously track more than one peak.
3. The procedure we use to evaluate C-18 reverse-phase columns is to determine the relative
number of peaks that are detected at a given slope sensitivity during the fractionation of an
aliquot of a large-scale tryptic digest of transferrin (3–5).
4. For high-sensitivity work, the baseline may be “balanced” (after running the first blank
run) by adding a small volume of 20% TFA (i.e., typically 10 to 100 µL) to either buffer A
or B as needed (5).
5. Because filtering HPLC solvents may result in their contamination (6), we recommend
they be made with HPLC-grade water and acetonitrile, and that they not be filtered prior
to use.
6. Provided the fractions are tightly capped within a few hours of collection (to prevent
loss of acetonitrile owing to evaporation), the acetonitrile is extremely effective at preventing
microbial growth and peptide loss owing to adsorption. Under these conditions,
we have often successfully sequenced peptide fractions that have been stored for longer
than a year.
7. Provided that samples of both the modified and unmodified protein are available,
comparative HPLC peptide mapping provides an extremely facile means of rapidly
identifying peptides that contain posttranslational modifications. In the case of proteins
that have been expressed in E. coli, the latter can often serve as the unmodified control,
since relatively few posttranslational modifications occur in this organism. Certainly, the
first attempt at comparative HPLC peptide mapping should be with enzymes, such as
trypsin or lysyl endopeptidase, that have high specificity, and the digests should be
separated using acetonitrile gradients in 0.05% TFA. Although elution position (as detected
by absorbance at 210 nm) provides a sensitive criterion to detect subtle alterations in
structure, the value of comparative HPLC peptide mapping can be further enhanced by
multiwavelength monitoring and, especially, by on-line or off-line mass spectrometry of
the resulting peptide -fractions (see Chapter 8, which details the off-line use of
MALDI-MS for analyzing peptides). If comparative peptide mapping fails to reveal any
significant changes, it is often worthwhile running the same digest in the pH 6.0 phosphate-buffered
system. At this higher pH, some changes, such as deamidation of asparagine
and glutamine, produce a larger effect on elution position than at pH 2.0, where
ionization of the side-chain carboxyl groups would be suppressed. Another possible reason
for failing to detect differences on comparative HPLC is that the peptide(s) containing the