JASMS 2008-3793
Supplemental Materials
To assess the need for fast elution of the peptides to reduce back exchange, we
tested a longer (15 min) gradient for both RPLC columns, shown in Supplemental Figure
1.
As expected, both columns provided more efficient separation with the longer
gradient, and the ProZapTM C18 column began eluting proteolytic fragments ~6 minutes
before the JupiterTM C5 column.
Supplementary Figure 2 (left) shows the deuterium incorporation for each of four
myoglobin fragments after 8 min HDX incubation.
Supplementary Figure 2 (right)
shows the deuterium uptake for myoglobin fragment 53-69 after each of several HDX
incubation periods. For both LC columns, the shorter gradient resulted in less back
exchange than the longer gradient. Measured deuterium uptake increased by ~15-50%
for the ProZapTM C18 column relative to the JupiterTM C5 column.
The increase in
deuterium uptake is especially dramatic for the shorter exchange periods (e.g., 1 min
and 8 min). Less back exchange for the short exchange periods should facilitate
identification of conformational differences for the fastest exchanging segments of a
protein.
Myoglobin and KIT cannot be fully deuterated, even after 56 hours of H/D exchange.
The data were used to compare deuterium incorporation following LC separations (but
not for absolute back-exchange calculations). Supplemental Figure 3 illustrates the
56-hour deuterated myoglobin and KIT deuterium uptake for identical fragments
following both separations. Deuterium incorporation (in percentage of total exchangeable
backbone amide hydrogens) was calculated for 16 myoglobin fragments (4 fragments
had two charge states to provide additional confirmation Figure 3 (top). On average,
~55% (58%, considering the 95% original deuterium) deuteriums were retained for
apomyoglobin fragments after ProZapTM C18 and ~41% (43%, considering the 95%
original deuterium) following the JupiterTM C5. The ProZapTM C18 column retained an
average of 34% (35%, considering the 95% original deuterium) more deuteriums than
the JupiterTM C5 column.
In Supplemental Figure 3 (bottom), KIT fragment 206-211 was ~92% deuterated after
ProZapTM C18 HPLC.
The ProZapTM column reduced the back exchange level almost
completely for the backbone amide hydrogens of that segment.
(Note that the
deuterated buffer contains only ~92% deuterium, so that the maximum deuteration level
cannot exceed 92%.) However, other fragments exhibited higher back exchange levels,
for at least two reasons. First, the whole protein (presumably, its highly folded and/or
hydrogen-bonded segments) was not completely deuterated after a 56-hour exchange
period. Second, peptide exchange/back-exchange rates are related to their primary
sequences.
Peptides eluted from the ProZapTM C18 column (on average, peptides
retained ~76% (83%, considering the 92% original deuterium) deuteriums) exhibited
~25% (26%, considering the 92% original deuterium) higher deuterium uptake relative
to the JupiterTM C5 column (peptides retained ~61% (66%, considering the 92% original
deuterium) deuteriums).
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Supplemental Figure Legends
Figure 1. Elution profiles for myoglobin fragments through HPLC ProZapTM C18 (top)
and HPLC JupiterTM C5 (bottom) columns, with a 15 min gradient. Peptides eluted from
~1.0-3.5 min from the ProZapTM C18 column and from ~7.0-9.2 min from the JupiterTM
C5 column. The ProZapTM C18 column also provided better separation of the proteolytic
peptides than the JupiterTM C5 column.
Figure 2. Left: Deuterium uptake for myoglobin peptides after 8 min of exchange. Right:
Deuterium uptake for myoglobin fragment 53-69 (triply charged, with 16 exchangeable
backbone amide hydrogens) after each of four exchange periods.
In all cases, the
ProZapTM column with a short gradient resulted in the highest deuterium uptake, i.e.,
minimum back exchange. Error bars represent triplicate result range.
Figure 3.
Deuterium uptake level for each of 16 maximally exchanged myoglobin and
KIT fragment peptides. Top: Deuterium uptake level for myoglobin fragments after the
two separations. Myoglobin was incubated in 95% deuterated buffer (pH meter reading
7.8) for 56 h at 25 ºC, quenched and digested for 2 min, and separated by ProZapTM C18
LC with a 1.5 min short gradient. Fast chromatography with ProZapTM C18 increased
deuterium incorporation by 34% relative to JupiterTM C5. Bottom: Deuterium uptake
level for maximally exchanged KIT fragments.
KIT was incubated in 92% deuterated
buffer (pH meter reading 7.5) for 56 h at 25 ºC, quenched and digested for 2 min, and
separated by ProZapTM C18 LC with a 1.5 min short gradient. The fast chromatography
with ProZapTM C18 increased deuterium incorporation by 25% relative to JupiterTM C5.
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