Chemoenzymatic enrichment of phosphotyrosine-containing peptides

Article abstract of DOI:10.1002/anie.200700633

(Chemical Equation Presented) Change the Tyr: A tyrosinase-assisted chemoenzymatic method was used to modify and enrich phosphotyrosine-containing peptides from a peptide mixture. This approach could be used to study protein tyrosine phosphorylation, and represents a promising alternative to immunoaffinity purification by using antiphosphotyrosine antibodies.

Full text of DOI:10.1002/anie.200700633

Angewandte
Chemie
DOI: 10.1002/anie.200700633
Protein Phosphorylation
Chemoenzymatic Enrichment of Phosphotyrosine-Containing
Peptides**
Shuwei Li* and Dexing Zeng
Protein phosphorylation on serine, threonine, and tyrosine
residues is a dynamic post-translational modification that is
involved in virtually all biological processes, including cell
growth, proliferation, and differentiation. Many serious
diseases such as cancer and diabetes are associated with
dysregulation of this event, indicating its critical roles in
multiple cellular-signaling pathways. Thus, improved methods
with which to detect and analyze phosphorylation sites have
always been sought to help us understand this important
modification.[1]
Although there are a variety of methods available, mass
spectrometry (MS) has recently become the primary choice
for the study of protein phosphorylation owing to its high
sensitivity and high throughput.[2] Proteins are digested to
peptides, which are then analyzed with MS to identify
phosphorylated residues. However, it remains technically
challenging for such efforts because of intrinsic difficulties
associated with protein-phosphorylation research.[3] For
example, the stoichiometric ratio of phosphorylation is
often low so that it requires enrichment of phosphorylated
peptides from a large excess of unphosphorylated background
peptides. The signal of phosphorylated peptides in MS is
usually suppressed owing to the negative charge of the
phosphate group. In addition, the assignment of phosphor-
ylation sites can be ambiguous, which is caused by the poor
quality of the fragmentation of phosphorylated peptides. As a
result, it is highly desirable that new techniques can be
developed to address these issues.
Because the phosphate group from phosphoserine (pSer)
and phosphothreonine (pThr) can be cleaved through a b-
elimination mechanism to produce an orthogonal double
bond under basic conditions, which can further react with a
thiol-containing compound, peptides bearing these two
phosphorylated residues can be modified with a biotin[4] or
other tags[5] for affinity enrichment. The removal of the
phosphate group from peptides also improves their ionization
and fragmentation in MS, thus enhancing the detection limit
and simplifying de nova sequencing. Unfortunately, phospho-
tyrosine (pTyr) is stable under the same conditions, making
this approach useless for the study of protein tyrosine
phosphorylation. Therefore, current approaches to study
tyrosine phosphorylation generally rely on immunoaffinity
enrichment of pTyr-containing tryptic peptides with immobi-
lized anti-pTyr antibodies, which are then sequenced directly
by liquid chromatography (LC)-MS/MS to identify the sites of
phosphorylation.[6] Even though these methods have been
demonstrated with success, the low sensitivity of phosphory-
lated peptides in MS has not been solved, making it a limiting
factor because protein tyrosine phosphorylation is a modifi-
cation with a low abundancy that only counts for 0.05–0.5%
of total phosphorylation.[7]
Herein, we reported a novel chemoenzymatic method
based on tyrosinase that could modify the pTyr residues with a
biotin tag containing a cleavable linker, allowing their
enrichment based on the biotin–avidin system and removing
the signal-suppressing phosphate group simultaneously.
Tyrosinases are a family of enzymes that widely exist in
bacteria, fungi, plants, and animals and that are involved in
melanin synthesis.[8] These enzymes are copper-containing
proteins that use molecular oxygen to catalyze the reaction of
tyrosine to ortho-quinone through an ortho-dihydroxyl phe-
nylalanine intermediate. ortho-Quinone is a highly active
molecule that can easily react with other species, such as thiol-
containing compounds.[9] Tyrosinase, however, can not oxidize
the tyrosine residues whose phenolic group is masked by post-
translational modification including phosphorylation and
sulfation. Herein, we show that one could take advantage of
this property of the enzyme to enrich pTyr-containing
peptides from a large excess of unmodified peptides.
Scheme 1 shows the involvement of guanidination of the
C-terminal lysine of tryptic peptides with methylisourea,
followed by the acetylation of nonphosphorylated tyrosines
with a mixture of N-acetylimidazole (NAI) and acetyl N-
hydroxysuccinimide ester (Ac-NHS). Under these conditions,
the C-terminal lysine would be transformed to homoarginine,
which could significantly improve its sensitivity in MS
detection. The N-terminal a-amine group would be acety-
lated as well. Next, the phosphate group on pTyr residues
could be cleaved with alkaline phosphatase. Finally, in the
presence of N-biotinyl-4-amino-2-methylbutan-2-yl 2-mer-
captoethylcarbamate (BAC), which is a bifunctional molecule
containing both a biotin and a thiol group joined by an acid-
labile linker, the newly exposed phenolic group would be
oxidized by tyrosinase to an ortho-quinone, which would be
expected to undergo rapid and efficient Michael addition to
form a thiol adduct. If successful, this method would result in
only peptides containing pTyr residues coupling with the
biotin tag, allowing them to be enriched by binding with
avidin–agarose. The enriched peptides could then be released
from the resin and treated with trifluoroacetic acid (TFA) to
[*] Prof. S. Li, Dr. D. Zeng
Center for Advanced Research in Biotechnology
University of MarylandBiotechnology Institute
9600 Gudelsky Drive, MD 20850 (USA)
Fax: (+1)240-314-6255
E-mail: liw@umbi.umd.edu
[**] We thank Wei-Li Liao andIllarion Turko for MS/MS experiments.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
nase could catalyze a broad spectrum of tyrosine-
containing peptides, suggesting that this
approach would potentially be able to label any
pTyr-containing peptides from a proteolytic pep-
tide mixture.
To demonstrate whether or not this approach
could enrich pTyr-containing peptides from a
large excess of nonphosphorylated peptide mix-
ture, we decided to test the idea with a tryptic
E. coli lysate spiked with a synthetic pTyr-con-
taining peptide (pep-3: H-FENIVSGpYAHK-
OH, calculated m/z 1529.6). As this E. coli lysate
was pretreated with alkaline phosphatase prior to
trypsin digestion, any peptides other than the
anticipated synthetic peptide after the enrich-
ment step would indicate nonspecific contami-
nation. This was a complex biological system, yet
it was also simple enough for us to evaluate and
optimize our protocols. We then followed our
procedure to treat this mixture and obtained a
strong peak in the mass spectrum whose m/z ratio
matched that of the modified peptide (Figure 1a–
b; pep-4: Ac-FENIVSGUYAHX-OH, Ac =
acetyl; U = quinone–thiol adduct; X = homoar-
Scheme 1. Tyrosinase-assisted assay for pTyr-containing peptides. Conditions:
1a) methylisourea, 1b) N-acetylimidazole and acetyl N-hydroxysuccinimide ester;
2) alkaline phosphatase; 3) tyrosinase in the presence of BAC; 4a) removal of excess
BAC reagent andavidin enrichment, 4b) TFA.
remove the bulky biotin tag, which would also remove acetyl
groups on the nonphosphorylated tyrosine residues.
ginine; calculated m/z 1624.7), indicating efficient enrichment
of pTyr-containing peptides and removal of the nonspecific
As the guanidination of the C-terminal lysine,[10] the
acetylation of tyrosine[11] (see also the Supporting Informa-
tion), and the acetylation of the N-terminal amine are all well-
established chemical reactions that can go to completion
rapidly and cleanly, the key step in this method is the
oxidation of tyrosine to ortho-quinone by tyrosinase. It is
essential to prove that tyrosinase can transform tyrosine
residues embedded in any peptide sequence into a quinone
adduct completely and specifically in the presence of thiol-
containing compounds. This includes two requirements:
1) other naturally occurring amino acids, except cysteine,
which is usually alkylated in proteomic assays, should not
interfere with this enzymatic reaction; 2) different sequences
of pTyr-containing peptides should only have a minimum
effect on the activity of tyrosinase. We therefore did a series of
experiments to test the properties of tyrosinase. First, we
prepared a peptide (pep-1: H-FNMGpYKWDHSYR-OH,
calculated m/z 1682.7) by standard solid-phase peptide syn-
thesis, which contained all active functional groups existing in
20 naturally occurring amino acids except cysteine. We then
incubated this peptide with tyrosinase and a large excess of
cysteine. Under these conditions, pep-1 was fully converted
into
the
peptide–cysteine
adduct
(pep-2:
H-
FNMGpYKWDHSUR-OH, U = quinone–thiol adduct, cal-
culated m/z 1817.7) quickly, indicating that tyrosinase could
completely oxidize tyrosine residues from peptide scaffolds
without modifying other amino acids such as methionine,
tryptophan, and pTyr. To examine if tyrosinase can oxidize
various peptides with different sequences, we developed a
high-throughput, fluorescence-quench-based activity assay
for tyrosinase (see the Supporting Information). After
measuring the activity of tyrosinase on several peptide
substrates with diverse sequences, we concluded that tyrosi-
Figure 1. a) The structure of pep-4, which is the modified form of pep-
3. The numbers indicate the m/z ratio for that particular segment.
b) MS of pep-4 after our chemoenzymatic treatment. c) MS fragmenta-
tion of pep-4. All y series fragments andselectedb series fragments
are labeled. Note: b and y are two series of ions obtained after
fragmentation of a peptide in MS/MS. Ir =% relative intensity.
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Chemie
background. To compare the performance of our method with
immunoaffinity purification, we followed a literature protocol
to purify pep-3 with an anti-phosphotyrosine antibody[12] from
the same sample and run a MS assay. Even though this
antibody-enriched phosphorylated peptide (pep-3) also gave
good signals, the signal-to-noise ratio of pep-3 was less than
that of pep-4, implying that our method would potentially be
more sensitive (see the Supporting Information). Finally, we
were also able to cleanly assign the sequence of pep-4 after its
fragmentation in MS/MS (Figure 1c).
This tyrosinase-assisted method for enrichment of pTyr-
containing peptides from proteolytic peptide mixtures would
represent a promising alternative to immunoaffinity purifica-
tion by using anti-pTyr antibodies. Despite our approach
involving multiple steps, the removal of negatively charged
phosphate groups, in combination with the guanidination of
the C-terminal lysine, could potentially enhance the sensitiv-
ity of detection and allow the unambiguous assignment of
phosphorylation sites on peptides. In addition, this approach
would enable us to incorporate various isotopic tags onto pTyr
residues for the quantification of tyrosine phosphorylation
from multiple samples.[13] We are currently optimizing our
protocol to further improve the detection limit of our method.
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Received: February 11, 2007
Revised: April 17, 2007
Published online: May 15, 2007
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Keywords: biotin · mass spectrometry · proteomics ·
.
tyrosinases · tyrosine phosphorylation
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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