Tags for labeling protein N-termini with subtiligase for proteomics

Article abstract of DOI:10.1016/j.bmcl.2008.08.044

The peptide ligase subtiligase, derived from subtilisin, has been employed in the identification of protein N-termini in complex mixtures. Here, the peptide ester substrates for the ligation reaction were optimized with respect to solubility, resulting in greater incorporation of the N-terminal tags. Additionally, the quantitation of the incorporated tags was explored, and a 'click' chemistry-based derivatization provided the ability to quantitate the tag to low nanomolar concentrations by sandwich ELISA. These new tags should expand the utility of subtiligase for the proteomic study of N-termini.

Full text of DOI:10.1016/j.bmcl.2008.08.044

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6000–6003
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tags for labeling protein N-termini with subtiligase for proteomics
*
Hikari A. I. Yoshihara, Sami Mahrus, James A. Wells
Departments of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, University of California, San Francisco, CA 94158-2330, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The peptide ligase subtiligase, derived from subtilisin, has been employed in the identification of protein
N-termini in complex mixtures. Here, the peptide ester substrates for the ligation reaction were opti-
mized with respect to solubility, resulting in greater incorporation of the N-terminal tags. Additionally,
the quantitation of the incorporated tags was explored, and a ‘click’ chemistry-based derivatization pro-
vided the ability to quantitate the tag to low nanomolar concentrations by sandwich ELISA. These new
tags should expand the utility of subtiligase for the proteomic study of N-termini.
Received 16 July 2008
Revised 11 August 2008
Accepted 12 August 2008
Available online 19 August 2008
Keywords:
Subtiligase
Peptide ligase
ELISA
Ó 2008 Elsevier Ltd. All rights reserved.
Click chemistry
CuAAC
Proteomics
Nitrotyrosine
The study of the proteome is aided by tools that can separate
specific subsets of proteins. For example, in studying post-transla-
tional modifications (PTM), methods for enriching phosphopro-
teins and phosphopeptides have been developed.1 One common
PTM in biology is the production of new N-termini by proteolysis;
another is the modification of N-termini through acetylation2,3 or
myristoylation.4 Several laboratories have described indirect
means for isolation or enrichment of N-terminal peptides. These
techniques include strong cation exchange chromatography of
tryptic peptides,5 and the chemical labeling of amine-containing
functionality in the proteome6 followed by either diagonal chro-
matography7 or affinity purification.8 Alternatively, our laboratory
has developed a direct enzymatic method to affinity tag and enrich
for the subset of proteins with free N-termini in order to character-
ize cellular substrates of proteolysis, or, more generally, character-
ize the ensemble of native N-termini. A recent study using this
method identified nearly three hundred caspase substrates and
their precise cleavage sites from apoptotic Jurkat cells.9
This method utilizes subtiligase, a variant of the protease subtil-
isin BPN’ that contains a cysteine residue in place of the active site
serine-221 and an alanine residue replacing proline-225.10 Unlike
the wild-type enzyme, the resulting double mutant has negligible
protease activity, yet it can efficiently acylate free N-termini of
peptides or proteins using ester substrates. The workflow for the
application of subtiligase to the study of the proteome is outlined
in Scheme 1. A complex mixture of proteins is N-terminally labeled
with subtiligase using a peptide ester containing a biotin affinity
tag and a TEV protease11,12 cleavage site. Tagged proteins are sep-
arated from the unincorporated tag and digested with trypsin.
Tagged N-terminal peptides are captured on avidin beads, cleaved
off the beads by TEV protease treatment, and analyzed by LC–MS/
MS. While approximately 80% of N-termini are blocked in mamma-
lian cells, mostly due to acetylation,13,14 certain populations of pro-
teins are believed to have a greater fraction of free N-termini.
These include signal-sequence-dependent proteins transported
across membranes, such as nuclear-encoded mitochondrial
proteins.15,8
While this approach is a powerful and sensitive tool for identi-
fying free protein N-termini and the precise sites of proteolysis, it
has limitations. Subtiligase will hydrolyze the ester substrate and
ligation yields vary depending on the accessibility and, to some ex-
tent, on the nature of the N-terminal residues.16 In principle,
increasing amount of peptide ester will increase the extent of the
ligation reaction by providing more ester for ligation before it is
consumed by hydrolysis. TEVest2 (1, Table 1), the prototypical
peptide ester, is somewhat insoluble in aqueous solution and can-
not be used at concentrations higher than 1 mM.
Here, we explored whether it would be possible to improve the
yield of the tagging reaction and increase proteomic coverage by
producing more soluble peptide esters. The general strategy in
these designs was to add basic or acidic residues such that the
net charge of the ester would be strongly positive or negative at
neutral pH. Some of the esters were conjugated to biotin using a
PEG-type linker instead of the two aminohexanoic acid residues
in 1. In addition, to facilitate the routine quantitation of the tagged
proteins in complex mixtures by sandwich ELISA, the incorporation
of 3-nitrotyrosine and propargylglycine residues into the esters
* Corresponding author. Tel.: +1 415 514 4498; fax: +1 415 514 4507.
E-mail address: jim.wells@ucsf.edu (J.A. Wells).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.044

H. A. I. Yoshihara et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6000–6003
6001
tion, showed improved solubility. However, a much greater in-
crease in solubility was observed with analogs that instead
contained additional arginine residues (3–5). We chose to incorpo-
rate D-arginine in order to preserve the integrity of the tag during
the trypsin digest step of the proteomic workflow.
Subtiligase site
A
B
Biotin-AhxAhxGGTENLYFQSY-glc-Y-NH2
TEV protease site
We also wished to produce peptide analogs that could be used
to introduce functionality for quantitation and labeling post-TEV
protease cleavage. Quantitation would be an important new fea-
ture allowing users to analyze how much labeling one has in the
sample prior to any further fractionation and LC–MS/MS. We chose
to test incorporating 3-nitrotyrosine since this single residue can
be used in very quantitative and sensitive ELISA assays.17 Peptides
containing this residue, however, were significantly less soluble.
Alternatively, we found that propargylglycine could be incorpo-
rated without loss in solubility. The terminal alkyne side chain of
propargylglycine provides a ‘click’-able handle for derivatization
with a variety of functional groups post-subtiligase labeling using
the copper-catalyzed alkyne–azide cyclization reaction18,19 with
an azide-bearing chemical tag. Ester 4 was thus designed to com-
bine some of the features of a highly soluble peptide ester and con-
tain a propargylglycine residue immediately N-terminal to the
ester group. Since non-b-branched hydrophobic residues at this
position are good substrates for subtilisin20,21 it was anticipated
that the propargyl side chain would be well tolerated. At this posi-
tion, the propargylglycine residue can function in two roles. First, it
can function as a tag for derivatization and quantitation, in
principle along the entire workflow and not only prior to the TEV
protease cleavage step as with 3. Second, as a non-natural amino
Protein
H2N
O
O
H
N
NH
2
biotin
ENLYFQSY
O
O
OH
O
H
Subtiligase
N
NH
2
HO
O
OH
biotin
ENLYFQSY
Trypsin
Protein
biotin
ENLYFQSY
avidin
Peptide K/R
Peptide K/R
Peptide
+
avidin biotin
ENLYFQSY
Peptide K/R
TEV
Protease
avidin
biotin
ENLYFQ
Peptide K/R
SY
LC-MS/MS
Scheme 1. (A) Structure of peptide ester 1 with ligation and TEV protease cleavage
sites annotated. (B) Proteomics workflow for N-terminal labeling using subtiligase.
was also investigated. The primary structures of the peptide esters
and their solubilities are listed in Table 1.
Ester 2, a derivative of TEVest2 (1) that contained an additional
four glutamic acid residues at the N-terminus of the peptide por-
Table 1
Peptide ester structures and solubilities
Ester
Sequence
Solubility (mM)
1
2
3
4
5
Biotin-AhxAhxGGTENLYFQSY-glc-Y-NH2
ꢀ1–2
ꢀ10
>40
>50
>40
Biotin-AhxAhx-EEEE-GGTENLYFQSY-glc-Y-NH2
Biotin-Linker-dRdRdRdR–prG-TENLYFQSY-glc-R-NH2
Biotin-Linker-dRdRdRdR-GTENLYFQS-prG-glc-R-NH2
Biotin-Linker-dRdRdR-prG-TENLYFQSY-glc-RR-NH2
O
O
O
O
NH
Biotin-Linker =
HN
N
H
N
H
O
3
Figure 1. Avidin blots of ligation reactions in Jurkat cell lysates. (A) Comparison of
peptide esters at 1 mM and 10 mM. The limited solubility of 1 precludes its use at
10 mM. (B) Lysates incubated with increasing concentrations (1, 2, 4, 7 and 10 mM)
Ahx = 6-aminohexanoyl
prG = L-propargylglycinyl
glc = glycoloyl
of peptide ester in the presence of 10 lM subtiligase.

6002
H. A. I. Yoshihara et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6000–6003
acid, it will produce unique a2 and b2 ions when tagged peptides
are fragmented during tandem mass spectrometry. Abundant a2
and b2 ions, corresponding to the residual serine and tyrosine res-
idues of the tag, are observed with a QqTOF instrument when ana-
lyzing proteomic samples labeled using ester 1 and are a hallmark
of tagged N-terminal peptides. The incorporation of propargylgly-
cine instead could provide an even more distinct signature.
The performance of the new esters was assessed by small-scale
ligation reactions with Jurkat cell lysates, which were then ana-
lyzed by SDS-PAGE and blotted with streptavidin to detect the
incorporated tag (Fig. 1). At 1 mM, all the esters show comparable
or modest improvements in labeling compared to 1. However, at
higher concentrations the more soluble penta-Arg containing es-
ters (3, 4, and 5) showed significantly higher levels of labeling.
Redistribution of some of the Arg residues from the N- to the C-ter-
minal side of the ester (compare 3 vs 5) did not significantly alter
their performance. Ester 4, which introduces a propargylglycine
residue directly adjacent to the ligated protein’s N-terminus, does
not appear to perform as well with respect to labeling as 3 or 5 but
is still significantly better than 1. We note that esters 3, 4, and 5
caused some protein precipitation in cell lysates. It is known that
proteins containing N-terminal polyarginine tags are less thermo-
stable,22 but we could prevent precipitation by the addition of so-
dium chloride, urea or guanidinium chloride.
A
As a model to test the performance of a quantitation strategy
involving derivatization of a propargylglycine residue (Fig. 2a),
tripeptides consisting of (Ac)GlyTyr(NO2)Ala, a PEG linker and an
azide group were prepared. The derivatization of the propargylgly-
cine residue of 3 pre-bound to neutravidin in 96-well plates pro-
ceeded efficiently under typical conditions (Fig. 2b). The large
excess (250 equivalents or greater) of the azide-bearing nitrotyro-
sine peptide appears to ensure the complete reaction of the avidin-
immobilized 3, as standard curves using peptides containing
nitrotyrosine residues and pre-derivatized peptides showed simi-
lar sensitivity compared to standard curves prepared by on-plate
derivatization. Using this assay, 3 can be reliably detected at
30 nM, a level of sensitivity that allows quantitation of ligated N-
termini to an estimated lower limit of 0.2% of free N-termini in a
eukaryotic cell lysate. In practice, the material analyzed would be
a complex mixture of proteins, with some fraction N-terminally la-
beled and any residual unreacted tag removed by gel filtration.
In summary, we have developed new peptide esters as subtilig-
ase substrates for use in the proteomic labeling of free N-termini.
By incorporating several additional arginine residues, the solubility
of the peptides is markedly improved, and the ligation reaction can
be driven further by the addition of more ester. Adding propargyl-
glycine residues into the peptide esters allows the quantitiation of
the labeled N-termini by sandwich ELISA following derivatization
with a nitrotyrosine and azide containing peptide. These features
should to expand the utility of subtiligase for the study of proteol-
ysis and other PTMs of protein N-termini.
tagged protein
avidin
avidin
NO2
avidin
avidin
HO
"Click”
O
O
H
N
NH2
N3
O
N
H
N
H
O
O
O
N
N
Tyr(NO2)
N
tagged protein
avidin
avidin
avidin
avidin
ELISA for Nitrotyrosine
α -Tyr(NO2)
HRP
Acknowledgments
N
N
Tyr(NO2)
We wish to thank Pete Wildes, Nick Agard and Emily Crawford
for helpful discussions. Financial support was provided by the San-
dler Foundation, NIH (RO1 GM081051), and the Hartwell Founda-
tion. HAIY was supported in part by a Stewart Trust Cancer
Research Award from the UCSF Cancer Center.
N
tagged protein
avidin
avidin
avidin
avidin
Supplementary data
Nitrotyrosine ELISA
B
50000
25000
0
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.044.
References and notes
1. Reinders, J.; Sickmann, A. Proteomics 2005, 5, 4052.
2. Bradshaw, R. A.; Brickey, W. W.; Walker, K. W. Trends Biochem. Sci. 1998, 23,
263.
3. Polevoda, B.; Sherman, F. J. Biol. Chem. 2000, 275, 36479.
4. Towler, D. A.; Gordon, J. I.; Adams, S. P.; Glaser, L. Annu. Rev. Biochem. 1988, 57,
69.
10
100
1000
Concentration 3 (nM)
5. Dormeyer, W.; Mohammed, S.; Breukelen, B.; Krijgsveld, J.; Heck, A. J. J.
Proteome Res. 2007, 6, 4634.
6. Enoksson, M.; Li, J.; Ivancic, M. M.; Timmer, J. C.; Wildfang, E.; Eroshkin, A.;
Salvesen, G. S.; Tao, W. A. J. Proteome Res. 2007, 6, 2850.
7. Gevaert, K.; Goethals, M.; Martens, L.; Van Damme, J.; Staes, A.; Thomas, G. R.;
Vandekerckhove, J. Nat. Biotechnol. 2003, 21, 566.
Figure 2. Quantitation of tagging by derivatizationand ELISA. (A) Tagged proteins are
captured on an analytical scale in neutravidin-coated 96-well plates. The propargyl-
glycine residue is derivatized using a copper(I)-catalyzed azide–alkyne cyclization
reaction and the incorporated 3-nitrotyrosine is quantitated using an antibody
conjugated to horseradish peroxidase (HRP). (B) ELISA using peptide ester 3.

H. A. I. Yoshihara et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6000–6003
6003
8. Timmer, J. C.; Enoksson, M.; Wildfang, E.; Zhu, W.; Igarashi, Y.; Denault, J. B.;
Ma, Y.; Dummitt, B.; Chang, Y. H.; Mast, A. E.; Eroshkin, A.; Smith, J. W.; Tao, W.
A.; Salvesen, G. S. Biochem. J. 2007, 407, 41.
9. Mahrus, S.; Trinidad, J. C.; Barkan, D. T.; Sali, A.; Burlingame, A. L.; Wells, J. A.
Cell 2008 (in press). doi:10.1016/j.cell.2008.08.012.
16. Chang, T. K.; Jackson, D. Y.; Burnier, J. P.; Wells, J. A. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 12544.
17. Ye, Y. Z.; Strong, M.; Huang, Z. Q.; Beckman, J. S. Methods Enzymol. 1996, 269,
201.
18. Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535.
19. Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am.
Chem. Soc. 2003, 125, 3192.
10. Abrahmsen, L.; Tom, J.; Burnier, J.; Butcher, K. A.; Kossiakoff, A.; Wells, J. A.
Biochemistry 1991, 30, 4151.
11. Carrington, J. C.; Dougherty, W. G. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3391.
12. Speers, A. E.; Cravatt, B. F. J. Am. Chem. Soc. 2005, 127, 10018.
13. Brown, J. L. Biochim. Biophys. Acta 1970, 221, 480.
20. Gron, H.; Meldal, M.; Breddam, K. Biochemistry 1992, 31, 6011.
21. Wells, J. A.; Cunningham, B. C.; Graycar, T. P.; Estell, D. A. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 5167.
14. Brown, J. L.; Roberts, W. K. J. Biol. Chem. 1976, 251, 1009.
15. Neupert, W. Annu. Rev. Biochem. 1997, 66, 863.
22. Fuchs, S. M.; Raines, R. T. Protein Sci. 2005, 14, 1538.