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Volume 8 | No. 13
Using PEPscreen™ to Study Protein Phosphorylation
and Kinase Activity
George Yeh, Ph.D., Product Manager, Sigma® Life Science
Stacey Hoge, Market Segment Manager, Sigma Life Science Custom Products
Protein phosphorylation events mediate key cellular signaling
pathways, regulating diverse processes such as proliferation,
metabolism, and apoptosis. In the human proteome, which numbers
up to 23,000 proteins, around 650,000 possible phosphorylation sites
have been predicted, and about 100,000 of those sites have been
confirmed as actual sites of protein phosphorylation.1 Protein kinases
(PKs) are one of the major classes of proteins that phosphorylate
substrates as part of the signaling cascade. PKs occur in substantial
numbers in various organisms, from predicted numbers of 518 in
humans2 up to 1,429 in rice.3
Because of the sheer scale of possible interactions between protein
kinases and potential targets (clients), establishing precise interactions
between specific PKs and particular sites is crucial to elucidate
related biological pathways. On a more technical level, highthroughput assays are needed to establish these valid kinase-client
interactions. Past methods have used low-throughput methods such
as radiolabeling or 2D-gel electrophoresis. More recent studies have
utilized peptide and protein arrays for higher throughput assays.1
Arrays have the disadvantage, however, that the conformations of the
proteins and peptides in the arrays are not native. Thus, interactions
between kinases and their substrates are not necessarily reflected
accurately. In addition, arrays do not reveal specific individual sites of
phosphorylation when a given peptide has multiple amino acids that
can be phosphorylated.
Mass spectrometry (MS) has emerged as a highly sensitive and specific
technology for probing protein phosphorylation.4 Professor Jay Thelen
of the University of Missouri has used Sigma-Aldrich’s PEPscreen
custom peptide libraries technology to develop high-throughput
tandem-MS assays for studying kinase phosphorylation of target
proteins. His group has developed their Kinase Client (KiC) assay to
probe kinase-mediated phosphorylation of multiple target peptides
in a single solution-phase reaction.5 The PEPscreen peptides provide a
convenient and cost-efficient source of custom-made peptide libraries
for such assays. These peptides are of sufficiently robust purity that
parent PEPscreen peptides and subsequently phosphorylated peptides
can be readily distinguished in LC-MS/MS analysis.
The group’s earliest report, by Huang et al., created a peptide
library consisting of 80 PEPscreen peptides from Sigma-Aldrich.2
These peptide sequences were principally derived from the serinecontaining peptides of three Arabidopsis thaliana proteins: pyruvate
dehydrogenase kinase (PDK, At3g06483), and two mitochondrial
pyruvate dehydrogenase complex (PDC) subunits, E1α-1 (At1g59900)
and E1α -2 (At1g24180). Their initial peptide cocktail contained 46
peptides used as substrates in a single kinase solution reaction.
The kinases used in the phosphorylation reaction were A. thaliana
PDK and an A. thaliana Ca2+-dependent protein kinase (CPK3,
At4g23650). The results showed that PDK phosphorylated a single
peptide, YHGHSMSDPGSTYR (underlined Ser, Ser292), under their assay
conditions. By contrast, PK3 did not phosphorylate any of the PDK
or E1α peptides. This demonstrated the precision of the assay under
conformational and thus relevant solution conditions. Focusing on the
YHGHSMSDPGSTYR peptide, the authors further studied the effects of
sequence variation around the primary site of phosphorylation, Ser292,
using an additional set of PEPscreen peptides. Additionally, the second
screen included a positive control peptide for CPK3, RASAIKALGSFASN,
which contains the phosphorylation site, Ser274, for plasma membrane
intrinsic protein 3. CPK3 phosphorylated only the RASAIKALGSFASN
peptide at the reported site.
In a study published in 2012, Ahsan et al. further probed the
YHGHSMSDPGSTYR peptide in terms of phosphorylation of Ser292
in the mitochondrial pyruvate dehydrogenase complex.7 This study
used a library of 59 PEPscreen peptides, where unlike the earlier study,
the sequences were all variations on that one peptide, to determine
exhaustively the effects of sequence variation around Ser292 on the
degree of its phosphorylation. The authors used recombinant PDK for
the phosphorylation reaction. In effect, this MS study offered a more
economical and faster alternative to a full-scale site-directed mutagenesis
study of full proteins with the corresponding sequence variations.
Your Biology Resource
Protein Phosphorylation and Kinase Activity | Volume 8 No. 13
Professor Thelen’s group has indicated up to 100 peptides can be
included in a single KiC assay.5 Using this reaction scale, their most
recent use of the PEPscreen technology involves a library of 377
peptide sequences of in vivo phosphorylation sites, to examine
phosphorylation by 77 different protein kinases from A. thaliana.8
Ahsan et al. chose the sequences from past identification of in vivo
phosphorylation sites in the developing seed of A. thaliana. Initial
screening revealed 23 proteins and 17 PKs of particular interest as
candidates for kinase-client pairing. The authors then focused on
one full-length protein, protein phosphatase inhibitor-2 (AtPPI-2), as
a particular client with potential roles in multiple signaling pathways.
As with the prior detailed investigations of the YHGHSMSDPGSTYR
peptide, this more recent study demonstrates greater efficiencies
in time and resources by focusing initially on target peptides for
phosphorylation, rather than trying to work on full-length proteins
with each of those 377 sequences.
Tandem MS continues to grow in importance in high-throughput
screening as an alternative to the use of arrays, and radiolabeled or
fluorescent substrates. Compared to arrays, tandem MS allows the
analysis of kinase-mediated phosphorylation in conformationally
relevant solution states of both kinase and client. Perhaps the most
important advantage of tandem MS over peptide arrays is the ability
to indicate phosphorylation site specificity in peptides with multiple
Ser, Thr, or Tyr residues. In this context, the PEPscreen custom peptide
libraries provide a promising avenue for investigating applications like
protein kinase activity and phosphorylation.
©2013 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC,
registered in the US and other countries. Where bio begins and PEPscreen are trademarks of Sigma-Aldrich Co. LLC.
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References
1. Zhang, H., and Pelech, S., Using protein microarrays to study
phosphorylation-mediated signal transduction. Semin. Cell Dev. Biol.,
23(8), 872-882 (2012).
2. Manning, G., et al., The Protein Kinase Complement of the Human
Genome. Science, 298(5600), 1912-1934 (2002).
3. Ding, X., et al., A Rice Kinase-Protein Interaction Map. Plant Physiol.,
149(3), 1478-1492 (2009).
4. Mann, M., et al., Analysis of protein phosphorylation using mass
spectrometry: deciphering the phosphoproteome. Trends Biotech.,
20(6), 261-268 (2002)).
5. Huang, Y., and Thelen, J.J., “KiC Assay - A Quantitative Mass Spectrometry-Based Approach for Kinase Client Screening and Activity
Analysis.” Quantitative Methods in Proteomics: Methods in Molecular
Biology, Vol. 893 (Springer Science), pp. 359-370 (2012).
6. Huang, Y., et al., A quantitative mass spectrometry-based approach
for identifying protein kinase clients and quantifying kinase activity.
Anal. Biochem., 402, 69-76 (2010).
7. Ahsan, N., et al., “Scanning mutagenesis” of the amino acid sequences
flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex. Front. Plant Sci., 3, Article 153 (2012).
8. Ahsan, N., et al., A versatile mass spectrometry-based method to
both identify kinase client-relationships and characterize signaling
network topology. J. Proteome Res., 12(2), 937-948 (2013).