Synthesis, characterization, and application of iodoacetamide derivatives utilized for the ALiPHAT strategy

Article abstract of DOI:10.1021/ja076849y

Hydrophobic tags, derived from iodoacetamide, were synthesized, characterized, and utilized to alkylate peptide E-76, a known inhibitor of coagulation factor VIIIa, and two other cysteine containing peptides. The electrospray response of each tagged pepti

Full text of DOI:10.1021/ja076849y

Published on Web 01/26/2008
Synthesis, Characterization, and Application of Iodoacetamide Derivatives
Utilized for the ALiPHAT Strategy
D. Keith Williams, Jr., Corey W. Meadows, Ibrahim D. Bori, Adam M. Hawkridge,
Daniel L. Comins, and David C. Muddiman*
Department of Chemistry and W.M. Keck FT-ICR Mass Spectrometry Laboratory, North Carolina State UniVersity,
Dabney Hall, Raleigh, North Carolina 27695-8204
Received September 13, 2007; E-mail: david_muddiman@ncsu.edu
Since the completion of the human genome project and with
subsequent advances in databases, mass spectrometry has emerged
as a leading technology in proteomics.1-3 To analyze proteomic
samples, cysteines are normally reduced and alkylated to eliminate
the protein’s tertiary structure.4-6 The alkylation step is utilized in
many chemical tagging strategies because the reaction is simple
and efficient.5 Hydrophobic tagging of large biomolecules (>500
Da) was first described by Null et al.7 One tagging strategy is the
ALiPHAT strategy (augmented limits of detection for peptides with
hydrophobic alkyl tags),8 which is a method previously developed
by Frahm et al. in which electrospray response of peptides was
increased via a cysteine specific hydrophobic tag. In the previous
study, peptides modified with one hydrophobic tag, 2-iodo-N-
octylacetamide, were demonstrated to have improved limits of
detection relative to peptides alkylated with iodoacetamide. Here
in, we report the synthesis, characterization, and application of four
new hydrophobic tags as well as the application of a previously
developed tag for the ALiPHAT strategy.8
The ALiPHAT application of these hydrophobic tags was
completed on peptide E-76 (Table 1, peptide 1), whose amino acid
sequence is shown in Figure 1, and two additional peptides util-
izing liquid chromatography Fourier-transform ion cyclotron
resonance mass spectrometry (LC-FT-ICR-MS). The E-76 peptide
was chosen because of its biological significance as a potent
inhibitor of coagulation factor VIIIa9 and because its amino acid
sequence contained cysteines. Iodoacetamide was the standard
alkylating agent to which the other tags were compared. The
reaction of iodoacetamide with cysteine groups results in a
carboxyamidomethyl (CAM) modification. The peptides modified
with the hydrophobic tags were combined with their CAM-modified
counterpart such that identical molar amounts of each were injected
on-column. Scheme 1 shows general reduction of protein and
alkylation reactions of the hydrophobic tags used in these experi-
ments.
Figure 1. The overlaid XICs of the CAM (green) and n ) 4 (red) modified
peptide are shown as well as their mass spectra. Denoted is the charge
state, theoretical m/z, and observed mass measurement accuracy. The
sequence of the E-76 peptide as well as a labeled MS/MS spectrum
demonstrating the n ) 4 tag does not fragment under conditions of CID
nor hinder MS/MS experiments are also shown.
Scheme 1. Reduction and Alkylation with Hydrophobic Tags
The ratio of peak areas from the extracted ion chromatograms
(XICs) describes both the chromatographic and electrospray ioniza-
tion consequences of addition of the hydrophobic tag. This ratio
was utilized to quantify the results described herein.7
E-76 peptide alkylated with 2-iodo-N-(4-phenylbutyl)acetamide
(Scheme 1, n ) 4) was observed to elute 6.05 min after the CAM-
modified peptide. This represents an increase in mobile phase B
by 8.5%, which demonstrates that this modified peptide is indeed
more hydrophobic than the CAM modified peptide. The overlaid
XICs of this and the CAM modified peptides are shown in Figure
1. The ratio of the area of these peaks demonstrates an improvement
of 429 for the peptide modified with 2-iodo-N-(4-phenylbutyl)-
acetamide versus the standard CAM-modified peptide. This hy-
drophobic tag provided the greatest improvement over the CAM
modification for the E-76 peptide.
Also shown in Figure 1 are mass spectra; one representing the
CAM-modified peptide and one representing the peptide modified
with 2-iodo-N-(4-phenylbutyl)acetamide which have theoretical
m/z’s of 1164.0202 and 1296.1140, respectively, in the 2+ charge
state. MS/MS data is shown for this modified peptide which
demonstrates that the tag itself does not fragment. This is important
Table 1. Investigated Peptide Sequences
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C O M M U N I C A T I O N S
evident by the increased retention time; however, the OCAM tag
does not improve the ESI response as well as the tags with the
phenyl group.
The final tag examined was 2-iodo-N-dodecylacetamide (Scheme
1, n ) 10), which generates a dodecylcarboxyamidomethyl
(DCAM) modification to cysteines. The DCAM-modified E-76
peptide led to an increase in retention time of 16.90 min versus
the CAM-modified peptide. This difference in retention time
represents an increase in mobile phase B by 25.9%. DCAM
modification of the E-76 peptide results in the most hydrophobic
of any of the tagged peptides observed in this study. The peak ratio
of the XIC between the DCAM- and CAM-modified peptide yielded
a result of 0.6.
Figure 2 demonstrates the relationship between the differences
in retention time between the different peptides (Table 1) with the
iodoacetamide derived tags and their CAM-modified counterpart.
The modifications with the phenyl terminal group yielded better
improvements when compared to the tags with only alkyl chains
for the E-76 peptide. 2-Iodo-N-dodecylacetamide performed the best
for peptides 2 and 3 and provided for electrospray response
improvements of 179 and 2441, respectively.
These hydrophobic tags have been applied to three peptides
whose electrospray response improvements are summarized in
Figure 2. The data, presented herein, clearly show alkylation of
the E-76 peptide, with the hydrophobic tags synthesized and
characterized in this study, can improve ESI response >400 fold
as well as provide for improved chromatographic behavior. Peptides
2 and 3 showed improvement in electrospray response for all
hydrophobic tags in comparison to their CAM-modified counterpart.
Furthermore, peptide 3 was able to achieve an improvement of
>2000-fold improvement over its CAM-modified counterpart!
These improvements in electrospray response come at essentially
no experimental cost since the alkylation step is facile and carried
out in nearly all bottom-up proteomic analyses. These benefits will
be able to aid in the investigation of cysteine-containing peptides
and proteins that have low electrospray response or concentration.
Figure 2. ESI response fold improvement vs change in retention time in
relation to the CAM-modified peptide. Tags are represented by point shape
and peptides represented by numbers corresponding to Table 1.
to note because if the tag were to fragment under conditions of
collision induced dissociation (CID), it would make the interpreta-
tion of MS/MS data much more difficult. Tandem MS data were
analyzed for all peptides with each of the tags discussed herein.
None of the tags were observed to fragment. The fragmentation
pattern remained the same between peptides modified with iodo-
acetamide and the new hydrophobic tags, which demonstrates that
the new tags do not adversely affect the CID mechanism for
fragmentation.
E-76 peptide alkylated with 2-iodo-N-benzylacetamide (Scheme
1, n ) 1) improved the chromatography and ESI response. Figure
2 shows the improvement of 69. Modification of the peptide with
this tag resulted in a shift in retention time by 2.85 min. This shift
in retention time is a result of the peptide being more hydrophobic
which causes it to elute in a concentration of mobile phase B
increased by 3.4% when compared to the CAM-modified peptide.
The improvement in chromatography is visible by a narrower peak
which results from the more hydrophobic modified peptide being
able to be captured more efficiently at the head of the column which
favors sample concentration prior to elution, as compared to the
CAM-modified peptide. This phenomenon is also observed with
the other tags examined in this study.
2-Iodo-N-(phenethyl)acetamide reacts with cysteines to create a
modification observed in Scheme 1 with n ) 2. This modified E-76
peptide resulted in a slightly more hydrophobic peptide than when
modified with 2-iodo-N-benzylacetamide. This increased hydro-
phobicity results in an increase in retention time of 3.57 min over
the CAM modified peptide. This difference in retention time
corresponded to the elution of this tagged peptide in mobile phase
with an increase of 4.5% B over the CAM-modified peptide. The
ratio of the peak areas demonstrated an improvement of 105 for
the modified peptide, shown in Figure 2.
As previously shown, alkylation with 2-iodo-N-octylacetamide
(Scheme 1, n ) 6) creates an octylcarboxyamidomethyl (OCAM)
modification to cysteines in a peptide or protein sequence.8 The
addition of the OCAM tag to the cysteines of E-76 improved both
the chromatography and electrospray response of the peptide which
is evident by the increase in peak area by 27 times versus the CAM-
modified peptide, shown in Figure 2. The difference in retention
time was 8.33 min which corresponds to an increase of 13.4% B
for the OCAM peptide to elute. The increase in hydrophobicity is
Acknowledgment. The authors would like to acknowledge
financial support by the National Institutes of Health (Grant R33
CA105295), the W. M. Keck Foundation, and North Carolina State
University.
Supporting Information Available: Synthesis and characterization
data for the hydrophobic tags. This material is available free of charge
via the Internet at http://pubs.acs.org.
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