Table 1. Bibliography of applications using peptide (micro)arrays for enzyme profiling and
substrate identification. The table lists aim of the studies, screening molecules, detection
methods, displayed peptide libraries, array preparation technologies, and each publication
citation. Entries were arranged according to the enzymatic reaction and within each reaction
type according to the year of publication. In the case of kinases entries were divided into
subgroups.
Aim
Screening
molecule
Detection
Library displayed on
peptide array
Techn
ology
Ref.
PKA, TRI, TRII
cytoplasmic
domain
 32P-ATP and
phosphorimaging
Combinatorial peptide
library
SODA
(1)
PKC, PKA, CK1
and CK2
 32P-ATP and
phosphorimaging
SPOT
(2)
Kinases: Substrate Identification
Determination of
substrate specificity
Determination of
substrate specificity
X ≠ Ser, Thr, Cys
23 Mammalian PKC
substrate peptides
and aa replacements (P,
K, L, E, A)
Determination of
substrate specificity
ANPK
 32P-ATP and
phosphorimaging
Kinase substrates
known from literature
SPOT
(3)
Substrate specificity
determination
PKA, PKG
 32P-ATP and
phosphorimaging
Combinatorial peptide
library and interative
deconvolution
SPOT
(4)
libraries:
XXX12XXX, X = mixture
of all 20 aa, 1,2 =
individual aa
Determination of
substrate specificity
Dyrk1A (R.
norvegicus) and
truncation variant
 32P-ATP and
phosphorimaging
Dyrk1A substrate
peptide
RRRFRPASPLRGPPK
and 300 aa substitutions
SPOT
(5)
Determination of
substrate specificity
CDPK-1 (from
maize seedlings)
 32P-ATP and
phosphorimaging
Peptide LARLHSVRER,
truncations and aa
substitutions (L, A, R, E)
SPOT
(6)
Determination of
substrate specificity
and inhibitor screen
PKA and PKG
 32P-ATP and
phosphorimaging
Combinatorial peptide
libraries and interative
deconvolution
SPOT
(7)
libraries:
XXX12XXX,
XXXRK12X,
XRKKK12X,
1RKKKKK2,
LRKKKKKH12,
12LRKKKKKH, 1,2 =
defined aa, X = mixture
of all aa except Ser and
Thr
Determination of
substrate specificity
NEK6 wt and
inactive variant
 32P-ATP and
phosphorimaging
710 peptides from
annotated human
phosphosites,
DIPP
(8)
DIPP
(9)
substitutional analysis of
GLAKSFGSPNRAY
Determination of
substrate specificity
p60c-Src
Fluorescein
labelled anti-pTyr
ab
Combinatorial library,
start: YIYGSFK, deletion
library, Ala scan,
positional scan
and combinatorial library
XXXXYXXXX, X ≠ Cys,
Tyr
Profiling of phosphospecific antibodies and
detection of kinase
substrates
CK2
 32P-ATP and
phosphorimaging
or fluorescein
labelled anti-pTyr
ab (Pt66, Sigma
Aldrich), anti- pSer
and pThr abs
2304 phosphorylated
and non phosphorylated
peptides from annotated
human phosphosites for
ab profiling and 11096
peptides covering the
cytoplasmic domains of
all human membran
proteins and 700
biotinylated peptides for
comparison of
phosphorylation on
microarrays and in
solution
DIPP
(10)
Determination of
substrate specificity for
substrate prediction
Abl
 32P-ATP and
phosphorimaging
1433 Random
sequences 13meric
peptides with fixed
tyrosine residue in the
middle position
DIPP
(11)
Determination of
substrate specificity
M.musculus and
H.sapiens TSSK3
32P-ATP and
phosphorimaging
200 Peptides
DIPP
(12)
Determination of
substrate specificity
KPI-2
33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
DIPP
(13)
species
Substrate specificity
determination and
comparison
Dyrk1A, Dyrk2,
Dyrk4
33P-ATP and
phosphorimaging
720 Peptides derived
from human
phosphosites
DIPP
(14)
Determination of
substrate specificity
MELK
32P-ATP and
phosphorimaging
192 Peptides comprising
in vivo phosphorylation
sites and peptides
containing deletions or
aa substitutions
DIPP
(15)
Determination of
substrate specificity
Dbf1-Mob1 (S.
cerevisiae) and
inactive variant
N305A
 32P-ATP and
phosphorimaging
Oriented peptide library
and interative
deconvolution libraries,
DIPP
(16)
DCSD
(17)
library:
MAXXXXRXXSXXXXAK
KK, proteome array
containing 4400 GST
proteins from S.
cerevisiae and peptide
microarray with 2296
annotated human
phosphosites
Kinase substrate
screening
Abl, Her2,
VEGFR2/KDR
Anti-pTyr ab (PY20,
Exalpha
Biologicals), sec.
ab Cy3 labelled
10000 member PNA
encoded peptide library:
F-Q-AA4-AA3-Y-AA2AA1-Ile-Lys
AA1, AA2, AA4: I, V, F, P,
R, D-P, S, D-V
AA3: I, V, A, P, R, K, -A,
S, P
Substrate specificity
determination
MPK1-3 (S.
lycopersicum) and
MPK6 (A.
thaliana)
 33P-ATP and
phosphorimaging
976 Annotated kinase
substrate consensus
sites
DIPP
(18)
Determination of
substrate specificity
Plk4/Sak (M.
musculus) wt and
inactive catalytic
domain
 32P-ATP and
phosphorimaging
DYDIPTTENLYFQ,
substitutional analysis of
MSYYHHHHHH,
substitutional scan of
RKKKSFYFKKHHH and
KSFYFKKHHH and 32
peptides from proteins
containing putative
phosphorylation sites
and variants of positive
peptides
SPOT
(19)
Determination of
substrate specificity
p60 c-Src, Abl and
cytoplasmic
domains of EphA4
32P-ATP and
phosphorimaging
Array containing 144 Tyr
kinase substrate
peptides, array
containing all
cytoplasmic Y ontaining
motifs from 2chimaerin, claudin-4 and
FGFR1 and array
containing systematic aa
substitutions of Dok6
Y220 and RasGAP Y386
derived peptides and
multiple substitutions
SPOT
(20)
Verification of the dual
specificity of CK2
CK2, CK2
 32P-ATP and
phosphorimaging
CK2 derived peptides
and sequences based
on S. cerevisiae Frp3
togehter with positional
scan
SPOT
(21)
Substrate specificity
determination
Protein kinase 7
(P. falciparum)
33P-ATP and
phosphorimaging
1176 Kinase substrate
peptides
DIPP
(22)
Use of peptide arrays
for prediction of kinase
substrate specificities
c-Raf, MAP3K8
 33P-ATP and
phosphorimaging
1176 Peptides
containing human
phosphorylation sites
and second array
containing 1024
peptides
DIPP
(23)
Substrate identification
FLT3, inactive
FLT3 K644A
PDGFR and
inhibitors STI571,
AG1269
32P-ATP and X-ray
film imaging at –70
°C
384 Peptides derived
from known
phosphorylation sites
DPCC
(24)
Substrate specificity
determination
p60c-Src, Abl,
EGFR, JNK1,
Erk2, p38, PKA
Phos-tag-biotin
followed by
fluorescence
labelled
streptavidin, Cy5
labelled anti-pTyr
ab
> 290 Tyr containing
peptides, > 1100 Ser or
Thr containing peptides
and new PKA
substrates:
RKRRGSLDESD,
RKRRASLDESD,
RKRRGSFDESD,
RKRRASFDESD,
TKRSGSVYEPL,
GAQAASPAKGE,
ALRRApSLGW,
ALRRASLGW,
ALRRAALGW
DIPP
(34)
Analysis if PknB can
phosphorylate human
peptides and substrate
PknB from S.
aureus
33P-ATP and
phosphorimaging
976 Disease related
human kinase
phosphorylation sites
DIPP
(26)
specificity
determination
Determination of
substrate specificity
followed by p53
homology screen
DAPK, Chk1
33P-ATP and
phosphorimaging
192 Kinase peptide
substrates
DIPP
(27)
Substrate specificity
determination
PKA isoforms
Tpk1 and Tpk2
from S. cerevisiae
33P-ATP and
phosphorimaging
SDLRRTS, LRRTSIIGTI,
GRQRRLSSLSEF,
QQTRRGSEDDTY,
LRRASLG and aa
substitutions
SPOT
(28)
SPOT
(29)
Kinases: Identification of phosphorylation sites in substrate proteins
Determination of
Lyn
Anti-pTyr ab (4G10, 23 PKC derived
phosphorylation sites
Upstate), HRP sec. peptides with central Tyr
Ab
in PKC
residue
 32P-ATP and
Edman sequencing
C-CAM-L derived
peptides, Glu variants
and Ala screen
SPOT
(30)
Gsk-3
-1
stimulated Hela
lysate
 32P-ATP and
phosphorimaging
p65 peptide scan and
systematic mutagenesis
of Ser, Thr and Tyr to
Ala
SPOT
(31)
Detection of
phoshorylation site in
AKAP-Lbc
PKA
 32P-ATP and
phosphorimaging
20mer peptides
encompassing Cterminus of AKAP-Lbc
SPOT
(32)
Determination of CK2
phosphorylation sites
in SSRP1
CK2
 32P-ATP and
phosphorimaging
SSRP1 derived peptides
containing Ser or Thr
and Ala variants
SPOT
(33)
Identification of
kinases responsible for
identified
phosphorylation sites
PKA, Akt, PKC,
PKC, PKC,
CK2, p38, Erk1,
Cdk5
32P-ATP and
phosphorimaging
190 Peptides
encompassing 95
phosphosites from MS
analysis and control
peptides
DIPP
(34)
Phosphorylation site
determination
Recombinant
human EphA4
receptor tyrosine
kinase
33P-ATP and
phosphorimaging
Overlapping 13meric
peptides corresponding
to the juxtamembran
region of hEphA4
SPOT
(35)
Phosphorylation site
determination
Recombinant
human EphA4
receptor tyrosine
kinase
33P-ATP and
phosphorimaging
Overlapping 13meric
peptides corresponding
to the juxtamembran
region of hEphA4
SPOT
(35)
Determination of
phosphorylation sites
in large cell-cell
adhesion molecule (CCAM-L)
PKC
Detection of
phosphorylation sites
on p65
(S. scrofa)
Identification of the
kinase responsible for
T840 phosphorylation
of GluR1
PKA, Akt1, PKC,
PKC, PKC,
PDK1, Cdk5, Raf1, p38, MEK1,
Erk1, Erk2, Rsk2,
JNK3, Gsk3,
CaMK2, CK1,
CK2, S6K, Rock2,
TBK1, p60 c-Src,
Fyn, Fes, Pyk2
32P-ATP and
phosphorimaging
NEAIRTSTLPRNSGA,
NEAIRTSVLPRNSGA
and peptides containing
consensus
phosphorylation sites
DIPP
(36)
Validation of new c-Src
substrates identified by
mass spectrometry
p60 c-Src
 32P-ATP and
phosphorimaging
312 Peptides
encompassing all Tyr
residues from 14
selected proteins and
Phe variants
DIPP
(37)
Phosphorylation site
determination
CaMK2
 32P-ATP and
phosphorimaging
23 Selected peptides
covering Ets-2 Ser or T
residues and positive
control pp28
SPOT
(38)
Validation of kinase
phosphorylation sites
Plk1, Aurora A
32P-ATP and
phosphorimaging
358 Identified spindle
phosphorylation sites
and VTELNDSRSECI
and aa scan
SPOT
(39)
Phosphorylation site
determination
PKA
32P-ATP and
phosphorimaging
20mer peptides
encompassing
intracellular loop of rat
cardiac Na+-Ca2+
exchanger NCX1, and
substitution analysis of
identified substrates
SPOT
(40)
DIPP
(41)
Kinases: enzymatic activities in cell lysates and kinome profiling
Comprehensive
PKA, lysate of
192 Kinase peptide
 33P-ATP and
description of
LPS and
substrates
phosphorimaging
phosphorylation events unstimulated
induced by LPS
PBMCs,
stimulation of PBMCs
MAPK and
inhibitors
PD98059,
SB203580
Analysis of kinase
activities in cell lysate
Lysates from M.
musculus MAFs
and M. musculus
MLCECs ± BMK1
knockout
 33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
species
DIPP
(42)
Fingerprinting effects
of DEX on T-
Lysate of anti-CD3
and anti-CD28 ab
stimulated CD4+
 33P-ATP and
phosphorimaging
1176 Peptides
containing kinase
substrate consensus
DIPP
(43)
lymphocyte kinome
cells treated ±
DEX
sequences
Comparison of kinome
profiles of Barrett’s
esophagus, normal
squamous esophagus
and normal gastric
cardia
Tissue lysates
 33P-ATP and
phosphorimaging
1176 Kinase specific
consensus sequences
DIPP
(44)
Investigation of the
effect of DEX on
adipocyte and Tlymphocyte kinome
Lysate of insulin
stimulated 3T3
adipocytes treated
± DEX and lysate
of anti-CD3 and
anti-CD28 ab
stimulated CD4+
cells treated ±
DEX
 33P-ATP and
phosphorimaging
1176 Peptides
containing kinase
substrate consensus
sequences
DIPP
(45)
Detection of kinase
activity in cell lysate
PKA, PKC, MCF-7
lysate incubated ±
Forskolin and H89
Biotinylated Phostag followed by
DyLight 647 or Cy3
labelled antistreptavidin ab
LRRAXLW, YIXGSFK,
LRVQNXLRRRR, X = S,
pS, T, pT, Y, F, A
DIPP
(46)
Analysis of kinase
activities in lysates
Lysates from 2
and 5 weeks old
A. thaliana and
from P. syringae
infected A.
thaliana
 33P-ATP and
phosphorimaging
192 Kinase peptide
substrates
DIPP
(47)
Screening of renal
kinase activities to
identify kinases
involved in angiotensin
II dependent
hypertensive renal
damage
Cortical tissue
lysates from
sprague dawley
rats and Ren 2
rats treated ±
ACEi
 33P-ATP and
phosphorimaging
> 1100 Mammalian
kinase substrate
sequences
DIPP
(48)
Tyrosin kinase profiling
in zebrafish embryos
Zebrafish embryo
lysate (1 day post
fertilisation) and
knockdown of Src
family kinases,
Fyn, Yes and
Wnt11, Fyn and
Yes kinase
Fluorescein
labelled anti-pTyr
ab (PY20, Exalpha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(49)
Tyrosine kinase
profiling in zebrafish
embryos
Zebrafish embryo
lysate 3 and 5
days post
fertilisation
Fluorescein
labelled anti-pTyr
ab (PY20, Exalpha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(50)
Determination of
kinase activities in cell
lysates to analyse
phylogenetic relations
of microarray
phosphorylation
patterns
Lysates from P.
pastoris, T.
aestivum, C.
albicans, A.
thaliana, F. solani,
D. melanogaster,
H. sapiens, M.
musculus, D.
discoideum and
 33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
species
DIPP
(51)
Effects of Pichinde
virus infection on
signalling pathways in
C. porcellus
macrophages
Cytoplasmic
macrophages
extract after mild
and lethal virus
infection (P2, P18)
1d and 6d post
infection
 32P-ATP and
phosphorimaging
1176 Peptides
containing human in vivo
phosphorylation sites
DIPP
(52)
Phosphorylation
profiling of A. thaliana
proteins
Cytosolic and
nuclear extracts,
cytosolic extracts
after stress,
SRPK4 (A.
thaliana), MPK3
(A. thaliana)
 32P-ATP and
phosphorimaging
47 Peptides contaning
known plant
phosphorylation sites
DIPP
(53)
Detection of kinase
activities in cell lysates
using SPR
p60c-Src, PKA,
EGFR,
MAPKAPK-2,
CaMK2, Akt,
p38, JNK1,
PKC, Erk2,
PKC, Abl, InsR,
Jak1, PKC and
inhibitors SU6656,
H-89 and A431
cell lysate ± NGF
stimulation and
SU6656 addition
pTyr detection:
anti-pTyr ab (Pt66,
Sigma Aldrich)
followed by SPR
26 Peptides containing
kinase consensus motifs
DIPP
(54)
Kinase profiling of
virion components
SARS coronavirus
lysate
33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
species
DIPP
(55)
Fingerprinting of
tyrosine kinase
activities
Lysates of
NUP214-Abl and
BCR-Abl
expressing Ba/F3
cells incubated ±
imatinib
FITC labelled antipTyr ab (PY20,
Exaplha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(56)
Fingerprinting of
tyrosine kinase
Lysates of human
dermal
FITC labelled anti-
144 Tyr kinase substrate
DIPP
(57)
pSer/pThr
detection: Phostag-biotin followed
by streptavidin,
anti-streptavidin ab
and SPR
activities
microvascular
endothelial cells
and of
hemangioma
endothelia cells
pTyr ab (PY20)
peptides
Analysis of the effect
of aspirin and
celecoxib on cancer
kinome
Lysates from
colon cancer cell
lines DLD1 and
HT29 treated with
aspirin or with
celecoxib
33P-ATP and
phosphorimaging
1176 Kinase substrate
peptides
DIPP
(58)
Fingerprinting of
tyrosine kinase
activities
Lysates of
androgensensitive prostate
carcinoma cell line
LNCaP
FITC labelled antipTyr ab (PY20,
Exaplha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(59)
Analysis of the
postsynaptic
phosphoproteome
PKA, CaMK2,
Erk2, Rock2,
Gsk3, Fes,
JNK3, Akt1,
PKC, PKC,
PKC, p70S6,
Rsk2, Erk1, Cdk5,
p38, CK1, CK2,
c-Raf, MEK1,
PDK1, TBK1, Fyn,
Pyk2
32P-ATP and
phosphorimaging
600 Peptides
corresponding to 300 M.
musculus in vivo
phosphorylation sites in
92 synaptic proteins
together with A, V or F
variants of the S, T and
Y residues and
additional peptides
containing kinase
consensus motifs and a
priming array containing
200 peptides from
multiple phosphorylated
sequences togehter with
A, V or F variants of the
S, T and Y residues and
pS, pT and pY
substitutions
DIPP
(60)
Kinome profiling for
putative target
detection and analysis
of differences in
primary and metastatic
UM lysates
Chondrosarcoma
lysates from 9
primary cell lines
and OUMS27,
CH2879, SW1353
and C3842 cell
lines and lysates
from 2 MSC
cultures and 5
colorectal
carcinoma cell
lines and inhibitors
PP1, PP2 and
PP3 and Mel270
cell lysate treated
 33P-ATP and
phosphorimaging
1024 Kinase substrate
peptides
DIPP
(61)
± p60 c-Src siRNA
Investigation of the
role of phosphorylation
in plant sugar
response
Lysates from A.
thaliana treated
with water,
sorbitol, glucose
or sucrose
32P-ATP and
phosphorimaging
1176 and 1024 Kinase
substrate peptides
DIPP
(62)
Tyrosine kinome
profiling
Uveal melanoma
(UM) cell lysates:
8 primary UM, 3
UM metastases, 3
fresh UM samples
and 3 liver
metastases
FITC labelled antipTyr (PY20,
Exalpha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(63)
Kinome profiling
Lysates from A.
thaliana fed
sucrose or sorbitol
32P-ATP and
phosphorimaging
960 Kinase consensus
sequences selected for
their importance in
mammalian signal
transduction and 1152
kinase substrate
peptides of mixed
species
DIPP
(64)
Detection of kinase
activities in cell lysates
p60 c-Src, cytoslic
and membrane
fraction of MCF-7
lysates, inhibitors
PP2 and SU6656,,
NGF, CHO and
A431 lysates and
lysates from M.
musculus brain
and skin
Cy5 anti-pTyr ab
840 Peptides
DIPP
(65)
Kinome profiling for the
detection of aberrant
kinase activities in
neoplastic tissues
29 pediatric brain
tissue lysates and
lysates from
Wilms’ tumors,
colon carcinomas,
normal kidney and
normal colon
Fluorescein
labelled anti-pTyr
ab (PY20, Exalpha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(66)
Kinome analysis of
monocytes
Lysates of
monocytes from
B. taurus treated ±
LPS
 32P-ATP and
phosphorimaging
298 Peptides containing
proposed B. taurus
phosphorylation sites
togehter with 2 negative
and 7 positive controls
DIPP
(67)
Fingerprinting tyrosine
kinase avtivities
Lysates of
leukemia samples
FITC labelled antipTyr ab
144 Tyr kinase substrate
peptides
DIPP
(68)
Kinome profiling and
B-cell lysates from
88 Systemic
33P-ATP and
1024 Kinase substrate
DIPP
(69)
biomarker identification
Lupus
Erythematosus
patents and from
72 healthy
controls
phosphorimaging
peptides
Kinome profiling of
osteoblast adhesion
MC3T3-E1 cell
lysate 2h after
adhesion
substrate
subjection and
control
33P-ATP and
phosphorimaging
1024 Kinase substrate
peptides
DIPP
(70)
Kinome profiling of
myxoid liposarcoma
Lysates of two
myxoid
liposarcoma cell
lines and primary
 33P-ATP and
phosphorimaging
1024 Kinase substrate
peptides
DIPP
(71)
cultures of four
myxoid
liposarcomas
Screening of kinase
activities in response
to Jasmonate and
salicylate treatment
Lysates of A.
thaliana incubated
with jasmonic acid
and/or salicylic
acid
 33P-ATP and
phosphorimaging
1178 Kinase substrate
peptides of mixed
species
DIPP
(72)
Tumor kinase profiling
to predict
chemotherapy
response
Biopsy lysates
from rectal cancer
patients ± sunitinib
Fluorescentlylabelled Anti-pTyr
ab
144 Tyr kinase substrate
peptides
DIPP
(73)
Fingerprinting tyrosine
kinase avtivities
Lysates from 4
different cell lines
and their matching
phosphatase and
tensin homolog
(PTEN)
knockdown
sublines
Fluorescentlylabelled Anti-pTyr
ab
144 Tyr kinase substrate
peptides
DIPP
(74)
Fingerprinting tyrosine
kinase avtivities
Lysates from
paediatric brain
tumor tissue
Fluorescentlylabelled Anti-pTyr
ab
144 Tyr kinase substrate
peptides
DIPP
(75)
Combinatorial peptide
library and interative
deconvolution
SPOT
(76)
Kinases: inhibitor identification and characterization
Identification of
Enzyme I of the
 32Ppeptides that inhibit
bacterial
phosphoenolpyruva
enzyme I of the
phosphotransferas te and
bacterial
e system
phosphorimaging
phosphotransferase
system
libraries:
XXXXXB1HB2XXXXX,
XXXXB1HB2XXXX,
XXXXXB1CB2XXXXX
B = defined, X =
randomized
Identification of
peptidic inhibitors
c-GMP dependent
kinase
32P-
autophosphorylated
PKG and
phosphorimaging
Combinatorial peptide
library and interative
deconvolution
SPOT
(77)
libraries:
XXX12XXX,
XXXRK12X,
XRKKK12X,
1RKKKKKH12,
12LRKKKKKH
1,2 = defined aa, X =
mixture of all aa except
Ser, Thr and
Ala screening of
selected peptides
Determination of Ki
values for inhibitors
p60c-Src and
inhibitors
quercetin,
tyrphostin, A47
and PP1
 33P-ATP and
surface plasmon
resonance (antipTyr ab, BD
biosciences)
YGEFKKK, FGEFKKK,
pYGEFKKK, YAAPKKK,
LRRASLG
DIPP
(78)
Investigation of
signalling pathways
influenced by
spongistatin 1
HUVEC lysate
treated ±
spongistatin 1,
combretastatin A4
phosphate or
vinblastine
 33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
species
DIPP
(79)
Prediction of kinase
inhibitor resistance in
tumors – profiling of
repsonse to MTKI of
27 cell lysates to
identify signature
peptides
HT29 lysate
transfected with
p60 c-Src construct
or empty vector
incubated ± PP2
or JNJ-26483327
(=MTKI), HCT116
and SKOV-3
lysate with p60 cSrc siRNA,
KatoII,SNU-5,
MKN45,
NSCLC.H441,
NCI-N87,
SNU484, NSCLCH322 and Caco2
lysates incubated
± JNJ-38877605,
FITC labelled antipTyr ab (PY20,
Exaplha
Biologicals)
144 Tyr kinase substrate
peptides
DIPP
(80)
EGF stimulated
lysates and
lysates from 8
xenograft tumors
incubated ± MTKI
Analysis of a putative
kinase inhibitor
Mesenchymal
stem cell lysates
incubated ±
inhibitor
PTK787/ZK22258
4
 33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
species, 12 control
peptides
DIPP
(81)
Analysis of the effect
of Pazopanib and
Lapatinib on A549
kinome
A549 cell lysates
treated ±
Pazopanib or
Lapatinib or both
HRP conjugate
anti-pTyr ab (R&D
Systems)
384 Peptides derived
from known
phosphorylation sites
DPCC
(82)
Effects of antiCD45RB ab on CD45
activity
PBMC lysates
after incubation
with anti-CD45RB
ab
 33P-ATP and
phosphorimaging
1176 Mammalian in vivo
kinase consensus
peptides
DIPP
(83)
Hydrogel based
microarray for
assessment of EGFR
activity and sensitivity
to inhibitors in cell
lysates of drug
resistant tumors
Lysates from NCIH23, NCI-H1650,
PC9, H1650-ER,
PC9-GR and
K562 lysates and
inhibitors erlotinib,
gefitinib, CL387,785, BIBW2992, CI-1033,
PKI-166
Anti-pTyr ab (4610
Upstate), sec. ab
HRP labelled
AEEEEYFELVAKKK
Diff
(84)
Identification of
signalling pathways
influenced by
flavopiridol
HUVEC lysate
treated with TNF ± kinase
inhibitor
flavopiridol
 33P-ATP and
phosphorimaging
1152 Kinase substrate
peptides of mixed
species
DIPP
(85)
 32P-ATP and
phosphorimaging
Combinatorial peptide
library and iterative
deconvolution:
XXX12XXX
SPOT
(86)
SPOT
(87)
Kinases: assay development/optimization
Method for substrate
PKA, PKG
specificity
determination using
peptide libraries
X = mixture of all 20 aa,
1,2 = individual aa and
peptides with different
length and Ala or D-Ala
variants as putative
inhibitors
Kinase assay on
PKA, PKC, CK1,
 32P-ATP and
Kinase substrate
peptides: RRASVA,
cellulose membranes
CK2
phosphorimaging
QKRPSQRAKYL,
DDDDEESITRR,
DDDSDDDAAA and Ser
to Ala variants, PKA
substrates with altering
length: AAAARASVA,
AARRASVA, RRASVA,
RRASVAAA,
RRASVAAAAA,
RRASCAAAAAA and 9
PKA substrate peptides:
LRRASVA, LRRASLG,
RRASVA, LRKASLG,
LHRASLG, LRHASLG,
LKRASLG, LARASLG,
LRAASLG
Method for substrate
specificity
determination using
peptide libraries
PKA
 32P-ATP and
phosphorimaging
Combinatorial peptide
library and interative
deconvolution
SPOT
(88)
libraries:
XXX12XXX, X = mixture
of all 20 aa, 1,2 =
individual aa
Demonstration of
applications for protein
and peptide
microarrays
p42 MAPK, PKA,
CK2
 33P-ATP and
phosphorimaging
Kemptide, Elk1, protein
kinase inhibitor 2
DIPP
(89)
Demonstration of
versatility of peptide
arrays for functional
assays
p60c-Src
 33P-ATP and
phosphorimaging
EEIYGEFF
DIPP
(90)
Development of an
antibody based
method to detect
kinase activity
p60c-Src, PKA
FITC-labelled
phospho-specific
abs
YIYGSFK, ALRRASLG,
YIYGpSFK,
ALRRApSLG
DIPP
(91)
Method for detection of
weakly interacting
protein-protein motifs
using paired peptides
Erk2
Anti-pElk1 (S383,
NEB) ab, HRP
labelled sec. Ab
KGRKPRDLELP,
FWSTLSPIAPR,
MNGGAANGRIL
SPOT
(92)
Validation of phosphospecific dye on solid
supports
Abl
ATP or -thio-ATP.
Phosphorylation
was detected with
Pro-Q Diamond
dye or
thiophosphorylation
was detected with
thiolreactive
Kemptide, Abl tyrosine
kinase peptide and
Ca2+/calmodulin kinase
substrate (glycogen
synthase 1-10) in 15
different dilutions on
polyacrylamide coated
CLPP
(93)
BODIPY 630/650
methyl bromide
reagent
slides
Method for
fluorescence detection
of phosphorylation on
peptide microarrays
Abl, PKA
ProQ Diamond
(Invitrogen)
Abl peptide, CaMK2
peptide, Kemptide
DIPP
(94)
Different methods on
peptide arrays for
kinase research
PKA, CK2, Gsk3,
PDK1, Tie2
 32P-ATP and
phosphorimaging
3 Arrays containing
either 710 human
phosphorylation sites or
2234 peptides ± priming
phosphorylations or
1394 kinase activation
loop derived peptides
and peptides covering
the cytoplasmic domain
of Tie2 as overlapping
peptide scan
DIPP
(95)
Method to evaluate
kinase activities on
peptide microarrays
using MALDI-TOF/MS
p60c-Src, PKA,
PKG, CaMK2,
CKI, Abl, Erk
MALDI-TOF/MS
IYGEFKKKC,
LRRASLGC,
RKRSRAEC,
KRQQSFDLFGC,
CKRRALpSVASLPGL,
IYAAPKKKL,
TGPLSPGPFGC
DIPP
(96)
inhibitors
Assay to detect
phosphorylation using
gold nanoparticle
probes
PKA and inhibitor
H89
Biotin-ATP followed
by avidin stabilized
gold particles, silver
enhancement and
resonance light
scattering
LRRASLG, LRRAGLG
DIPP
(97)
Development of a
microarray based multi
analyte assay which
allows incubation with
different enzymes in
one experiment
Abl, p60 c-Src, CK1,
Erk1, PKA, PKC,
PKG and
inhibitors Gleevec,
Gö6850 and
lysate from K562
cells that
overexpress BcrAbl ± Gleevec,
peroxide vanadate
and calynuclin A
treatment
MALDI-TOF/MS
AIYAAPFKKGC,
EEIYGEFE, AKKKC,
CKRRALpSVASLPGL,
TGPLSPGPFGC,
LRRASLGC,
AKIQASFRGHMARKKG
C, RKRSRAEC,
AIpYAAPFKKGC,
LRRApSLGC
DIPP
(98)
Method for detection of
phosphorylation on
peptide microarrays
PKA
 32P-ATP or
biotinylated Zn2+
chelate compound
followed by
streptavidin and
SPR, anti-pSer
CGGLRRASLG, Ala
substitution and Ser
phosphorylated,
CGIYGEFKKK and Tyr
phosphorylated
DIPP
(99)
(PSR-45, Sigma
Aldrich) or anti-pTyr
(PT-66, Sigma
Aldrich) ab followed
by SPR
Kinase inhibition assay
using sol-gel derived
multicomponent
microarray
PKA and inhibitors
H7, H89
ProQ Diamond
(Invitrogen)
Kemptide
Diff
(100)
Method for screening
of kinase – chemical
interaction using
aerosol deposition of
nanodroplets
containing the
biologically active
compound
p60c-Src, B-Raf
(V599E), KDR,
Met, Flt3 (D835Y),
Lyn, EGFR,
PDGFR, Tie2
and inhibitors
staurosporine,
PP2, GW5074,
KDRI and PDGRF
1-1
Anti-pTyr ab
(pTyr100, Cell
signalling), sec.
Alexa 555 ab
Poly(E4-Y)10 peptide
DIPP &
SPNS
(101)
Microfluidic peptide
microarray applications
PKA
ProQ Diamond
(Invitrogen)
R(R/K)XSLG, X = any
aa and pY, A and F
variants of S
LDPS
(102)
Method for employing
kinase inhibition based
on labelling
phopshorylation events
using nanogold
particles
PKA and
Biotin-ATP,
detection using
avidin stabilized
gold nanoparticles
followed by silver
enhancement and
resonance light
scattering
LRRASLG, LRRAGLG
DIPP
(103)
Solution phase kinase
assay
CK1, Plk3, Akt1,
SGK1, PKC,
PKCz, PKA,
Aurora A, Aurora
B, Chk2, CaMK2,
MK2, Chk1, Cdk2,
Cdk5, p38, CK2,
Abl, Lck, p60c-Src,
EphB2, VEGFR2,
Kit, Flt3, Jak2,
EGFR and
inhibitors PKI,
SrcI, TBB,
staurosporine
Fluoresceinylglycin
e amide followed
by anti-fluorescein
ab (Roche),
biotinylated sec. ab
and strepatavidinAlexa555 or antipTyr-biotin ab
(Invitrogen)
followed by
streptavidin
phycoerythrin
900 Peptide substrates
encoded by an
oligonucleotide
sequence composed of
annotated kinase
substrate sequences
and random sequences
DCSD
(104)
Fluorescence based
assay for
phosphorylation
detection
PKA, Akt1 and
inhibitor
staurosporine
-elimination
followed by Michael
addition of thiol
containing
RPRAASF, LRRASLG
SPOT
(105)
PKA inhibitors
H89, HA1077,
KN62, mallotoxin
tetramethylrhodami
ne
Protocol for
phosphorylation site
determination
Different human
kinases
33P-ATP and
phosphorimaging
or ProQ Diamond
followed by
fluorescence
reading
3280 Peptides covering
all human peptidylprolyl-cis/transisomerases and 17181
peptides covering the
CMV proteome
DIPP
(106)
Protocol for kinome
profiling using plant
tissues, biopsies, cells
and purified kinases
Plant tissues
lysates, biopsie
lysates, cells
lysate and purified
kinases
 33P-ATP and
phosphorimaging
1176 Kinase substrate
peptides from different
organisms and a second
arrays contaning 1024 in
vivo phosphorylation
sites
DIPP
(107)
Kinetic studies of PKA
catalyzed
phosphorylation in real
time on a peptide array
PKA and inhibitors
staurosporine,
AMP-PNP and
PKA inhibitor
peptide
Anti-phospho ab,
sec. fluorescently
labelled ab allowing
real time detection
of fluorescence
using a CCD
camera
140 Peptides derived
from human
phosphorylation sites
together with 4
phosphopeptides and
detailed phosphorylation
analysis of the peptide
EILSRRPSYRKIL and
kinetic analysis of CREB
peptide and Kemptide
(LRRASLG)
DIPP
(108)
Assay to identify
bisubstrate inhibitors
PKC, 
Anti-pSer, anti-pThr
ab (Cell signalling
#2351, 9611,
9391), sec. ab
FITC labelled
144 Ser/Thr Kinase
substrate peptides
DIPP
(109)
Kinase assay using
Raman spectroscopy
and gold nanoparticle
probes
PKA and inhibitors
H89, HA1077,
KN62
Biotinylated antipSer ab (Sigma
Aldrich) followed by
gold nanoparticles
and silver
enhancement
CGGALRRASLG,
CGGALRRAGLG
DIPP
(110)
Assay for the
development of a
depigmenting assay
PKC and
inhibitors TMP
and
bisindoylmaleimid
e
Anti-pSer/pThr ab,
sec. ab Cy5
labelled
30- and 10-meric
Peptides as MBP fusion
protein derived from
tyrosinase
Diff
(111)
Protocol for
measurement of
kinase activities in cell
lysates
A431 lysates ±
SU6656
incubation, HepG2
lysates and
lysates from M.
musculus tumors
after A431 cell
Phos-tag-biotin
followed by Dylight
649 conjugated
streptavidin or Cy5
labelled anti-pTyr
ab (Amersham)
Array containing 804
peptides and second
array containing
peptides EEIpYGEFD,
EEIYGEFD, EEIFGEFD
DIPP
(112)
inoculation
Protocol for
determination of
kinase activities in cell
lysates
HEK293 lysate
ProQ Diamond
(Invitrogen)
4198 Peptides
representing overlapping
peptide scans covering
all human peptidylprolyl-cis/transisomerases
Kinases: peptide immobilization and array fabrication optimization
Development of
p60c-Src, PKA
FITC-labelled
YIYGSFK, ALRRASLG,
approaches for site
phospho-specific
KGTGYIKTG,
specific immobilisation
abs
YIYGpSFK,
of peptides
ALRRApSLG,
KGTGpYIKTG
DIPP
(113)
DIPP
(114)
Review: peptide array
applications
c-Abl
 32P-ATP or
fluorescencelabelled anti-pTyr
ab (PY20)
720 peptides from
annotated human
phosphosites
DIPP
(115)
Immobilization
strategies for peptides
on peptide microarrays
PKA, p60c-Src
FITC-anti-pTyr and
FITC anti-pSer ab
(Sigma Aldrich)
YIYGSFK, ALRRASLG
DIPP
(116)
Development of an
oriented peptide library
approach
PKA, Cdc15 (S.
cerevisiae), ab
MPM2, 3F3/2 and
PY20 ab
 33P-ATP, antiHRP sec. Ab
Binding libraries:
AXXXXpS/pTXXXXA,
AXXXXpYXXXXA,
AXXXXXpS/pTQDPFXX
A, X ≠ Cys
SPOT
(117)
Kinase library:
AXXXXXS/TXXXXA, X ≠
Ser, Thr, Cys
Strategy for making
peptide arrays using
protein-peptide fusions
Abl, PKA
Cy3/Cy5 labelled
anti-pSer and pTyr
ab (Sigma Aldrich)
Leptin-Kemptide, malic
enzyme-Kemptide,
Leptin-Abl peptide, malic
enzyme-Abl peptide
DIPP
(118)
Immobilization using
poly(dT)-modified
peptides
p60c-Src,PKA and
inhibitor AG213
Anti-p-GFAP ab
(YC10 Medical and
Biochemical
Laboratories) and
anti-pTyr ab (PY20,
Exalpha
Biologicals)
IYGEFKKK, IAGEFKKK,
RRRVTSAARRS,
RRRVTAAARRS
DIPP
(119)
Assay for
phosphorylation site
detection using gold
nanoparticle based
electrochemical
p60 c-Src, PKA and
inhibitors ellagic
acid, PP2, PP3,
genistein,
herbimycin A
Biotin-ATP followed
by streptavidin
coated gold
nanoparticles and
electrochemical
Raytide, Kemptide
DIPP
(120)
detection
detection
Optimized surface
chemistry for peptide
immobilisation for
phosphorylation
analysis using different
linker moieties
p60c-Src, PKA
 33P-ATP or SPR
imaging using Zn2+
chelate compound
CGIYGEFKKK,
CGGLRRASLG and
modifications
DIPP
(121)
Microarray assembly
by accoustic
dispensing
Pim1 and inhibitor
hb1277
32P-ATP and
phosphorimaging
KKRNRTLTK
SPNS
(122)
Regeneration of
phosphorylated SPOT
membranes
Potato acid
phosphatase
32P-ATP and
phosphorimaging
Kinase substrate
peptides: RRASVA,
QKRPSQRAKYL,
DDDDEESITRR,
DDDSDDDAAA and S to
A variants, PKA
substrates with altering
length: AAAARASVA,
AARRASVA, RRASVA,
RRASVAAA,
RRASVAAAAA,
RRASCAAAAAA and 9
PKA substrate peptides:
LRRASVA, LRRASLG,
RRASVA, LRKASLG,
LHRASLG, LRHASLG,
LKRASLG, LARASLG,
LRAASLG
SPOT
(87)
PTP substrate
specificity
determination
PTP1B, PTP,
Anti-pTyr ab
mixture (4G10,
upstate, PY20 and
PY69, Transduction
Laboratories, pTyr100, Biolabs), HRP
sec. Ab
AABX1pYX2BAAA, B =
mixture of all 20 aa, X1,
X2 = individual aa, Tie2
peptides
VFSKYpYTPVLA,
VFSKYpYpTPVLA,
STAT5 peptide
MAIESLNYSVYTTNS
and pY, pS, pT variants
and IR
autophosphorylation
sequence with all
combinations of pY
SPOT
(123)
Substrate identification
GST-fusion of
substrate-trapping
mutants of PTPH1, TCPTP, SAP1, and PTP-1B
Radioisotopic
labelling of GSTfusion proteins by
PKA mediated
phosphorylation
followed by
7 Peptides derived from
cytoplasmic region of
GHR in nonphosphorylated and
tyrosine phosphorylated
form
SPOT
(124)
Phosphatases
phosphorimaging
Method for detecting
weakly interacting
protein-protein motifs
using paired peptides
PTP1B
Anti-pTyr ab
(Upstate), HRP
sec. Ab
IR autophosphorylation
site IYETDYpYRKGG
and overlapping
peptides covering the
cytoplasmic sequence of
IR
SPOT
(92)
Protocol for SPOT
applications for PTPs
PTP1B
Anti-pTyr ab
mixture (4G10,
upstate, PY20 and
PY69, Transduction
Laboratories, pTyr100, Biolabs), HRP
sec. Ab
IR peptide
MTRDIYETDYYRKGG
and pY substitutions and
SPOT double synthesis
covering the cytoplasmic
IR domain as
overlapping peptide
scan togehter with the IR
autophosphorylation
peptide IYETDpYRKGG
SPOT
(125)
Phosphatase profiling
using cell lysates
K562 cell lysates
treated ± Gleevec,
peroxide
vanadate,
calynulin A
MALDI-TOF/MS
AIYAAPFKKGC,
EEIYGEFEAKKKC,
CKRRALpSVASLPGL,
AIpYAAPFKKGC,
LRRApSLGC
DIPP
(98)
Construction of pTyr
microarrays for
phosphatase
specificity
determination
PTP1B, PTPµ
Fluorescence
imaging
subsequent to
treatment with Cy5
labelled anti-pTyr
ab
48 pTyr-containing
peptides
DIPP
(126)
Assay for substrate
specificity
determination
Lambda
Phosphatase, AP,
PP1, PP2B, PP2A
± Pin1
ProQ Diamond
(Invitrogen)
87 phosphopeptides
derived from know
phosphorylation sites
DIPP
(127)
Peptide immobilization
by click sulfonamide
reaction
PTP1B
Anti-biotin-strepCy5
PDVLEYpYKNEHAK,
RDINSLpYEVSRMY
DIPP
(128)
Effects of antiCD45RB ab on CD45
activity
PBMC lysates
after incubation
with anti-CD45RB
ab
33P-ATP and
phosphorimaging
1176 Mammalian in vivo
kinase consensus
peptides
DIPP
(83)
Identification of
physiological subtrates
GST-fusion of
substrate-trapping
mutant of human
Density-Enhanced
Phosphatase
(CD148)
Fluorescence
imaging
subsequent to
incubation with Cy5
labelled anti-GST
ab
6057 pTyr-containing
peptides derived from
human phosphorylation
sites and 343 controls
DIPP
(129)
Substrate specificity
determination,
identification of specific
substrates by dualcolor readout
Cy3 or Cy5
labelled substratetrapping mutants
of PTP-1B,
TCPTP, SHP-1,
SHP-2, and
LMWPTP
Fluorescence
imaging
144 Putative Tyr
phosphatase peptide
substrates
DIPP
(130)
Substrate specificity
determination
PTP1B, TCPTP,
SHP1, SHP2,
SHP1 and SHP2
catalytic domain,
LMWPTP
ProQ Diamond
(Invitrogen)
144 Putative Tyr
phosphatase peptide
substrates
DIPP
(131)
Identification of
physiological subtrates
GST-fusion of
substrate-trapping
mutant of human
PTP-1B
Fluorescence
imaging
subsequent to
incubation with Cy5
labelled anti-GST
ab
6057 pTyr-containing
peptides derived from
human phosphorylation
sites and 343 controls
DIPP
(132)
Lysin Methyl-Transferases (PKMTs)
Determination of the
contribution of aa for
substrate recognition
G9a
3H-AdoMet
and
tritiumimaging
Substitutional analysis of
a peptide covering the
first 21 aa of H3 (all 20
aa and modified aa) and
45 peptides from
putative new substrate
proteins
SPOT
(133)
Substrate specificity
determination
Dim-5 (N. crassa)
3H-AdoMet
and
tritiumimaging
Overlapping peptide
scan of the first 21 aa of
H3 and K9A variant and
substitutional analysis
SPOT
(134)
Substrate specificity
determination and
identification of novel
in vivo substrates
Set7/9
3H-AdoMet
- 420 peptides
representing
substitutional analysis of
histone H3 tail 1-21
SPOT
(135)
and
tritiumimaging
&
DPCC
- 133 predicted peptides
derived from 118
different proteins
- 216 peptides derived
from histone H3
featuring 18 different
posttranslational
modifications
Peptidyl-Prolyl-cis/trans-Isomerases
Identification of
Human Pin1
Sequential electroblotting to PVDF-
Peptide scan of human
Cdc25C including
SPOT
(136)
substrates
membranes,
incubation of
PVDV-membranes
with anti-Pin1 ab
followed by
secondary ab
phosphorylated S/T-P
moieties
Determination of
substrate specificity
Trigger factor (TF)
(E. coli) and TFfragments
Sequential electroblotting to PVDFmembranes,
incubation of
PVDV-membranes
with anti-TF ab
followed by
fluorescence
labelled secondary
ab
Peptide scans (13meric
peptides overlapping by
10 residues) of E. coli
proteins: elongation
factor Tu, methionine
biosynthesis enzyme,
isocitrate
dehydrogenase,
glutamine-tRNAsynthetase, alkaline
phosphatase, galactosidase, galactose
binding protein), L2 (a
ribosomal protein),
lambda cI, pro-outer
membrane protein A,
sigma 32, SecA,
dehydrofolate reductase
and yeast cytochrome
B2, F1- and Su9ATPase subunits,
Photinus pyralis
luciferase, RNaseT1
from Aspergillus oryzae
and human PrP (prion
protein)
SPOT
(137)
Determination of
substrate specificity
Trigger factor and
DnaK (E. coli)
Sequential electroblotting to PVDFmembranes,
incubation of
PVDV-membranes
with anti-TF ab
followed by
fluorescence
labelled secondary
ab
Peptide scans (13meric
peptides overlapping by
10 residues) of E. coli
proteins: elongation
factor Tu, methionine
biosynthesis enzyme,
isocitrate
dehydrogenase,
glutamine-tRNAsynthetase
SPOT
(138)
Identification of Pin1
inhibitors
Catalytic domain
of human Pin1
Sequential electroblotting to PVDFmembranes,
incubation of
PVDV-membranes
with anti-Pin1 ab
followed by
secondary ab
1000 pentapeptides of
general structure AcXaa-pThr-Yaa-ZaaGln(linker) with Xaa and
Zaa = Phe, Abz, Bip,
Bth, Cha, Dbg, Nal,
tBuGly, tBuPhe, Thi
SPOT
(139)
Yaa = MeAla, MePhe,
Pip, Pia, 4Pip, Thz, Tic,
Pro, Acc, Amb (all
possible combinations)
Identification of
substrates
Human Pin1
ProQ Diamond
subsequent to
phosphatase
treatment
87 phosphopeptides
derived from know
phosphorylation sites
DIPP
(127)
Arginin Methyl-transferases (RMTs)
Substrate screening
RMT1, CARM1
3H-AdoMet
and
tritiumimaging
36 PABP1 and H3
derived peptides
containing central Arg
SPOT
(140)
Solution phase assay
with tagged substrates
allowing selective
capturing
Protein arginine
methyl-transferase
1 (RMT1)
MALDI-TOF/MS
GGRGGFGC
DIPP
(141)
Consensus motif
determination
Sumo1 and Hela
cell lysate
Anti-Sumo1 ab
followed by IgG
biotin and antistreptavidin-HRP
ab
10 RanGAP1 derived
peptides containing
Sumo consensus motifs
and control peptides
containing R instead of
K for assay optimization,
peptides covering 11
Sumo targets and
negative control and
array containing 640
peptide library for
consensus motif
determination
DIPP &
Diff
(142)
Determination of
SUMOylation sites
SUMO kit (Biomol)
Anti-Sumo1 ab
Overlapping peptide
scan of PDE4B2, C2,
A4, D5 and alanine scan
of putative PDE4D5
sites
SPOT
(143)
Ubiquitination kit
(Biomol)
Anti-ubiquitin ab
Overlapping PDE4D5
peptide scan, 6 arrestin
2 derived peptides
SPOT
(144)
ppGalNACT
isoform T2
MALDI-TOF/MS
GAGAPGPTPGPAGAG
K,
GAGAPGPSPGPAGAG
K, GAPGPTPGPAGK,
AcPTPGPAGK,
DIPP
(145)
SUMO-Transferases
Ubiquitin-Transferases
Determination of
ubiquitinylation target
sites
Glycosyl-transferases
Assay for monitoring
O-glycosylation
GAPGSTAPPAGK,
GAHGVTSAPAGK,
GAAPDTRPAAGK,
YSPTSPSKR,
PTTDSTTPAPTTK,
DDSIEGSGGR
O-glycopeptide library
production for disease
associated
autoantibody
screening and
substrate specificity
comparison
GalNAc-T1 and
T2
MALDI-TOF/MS,
lectin reactivity and
mabs 5E5 and 2D9
VTSAPDTRPAPGSTAP
PAHG ± GalNAc,
PTTTPITTTTTTVTPTPT
GTQTPTTTPISTTC,
CPPTPSATTPAPPSSS
APPETTAA,
LSESTTQLPGGGPGCA
,
MEELGMAPALQPTQG
AMPAF,
PRFQDSSSSKAPPPSL
PSPSPLPG,
WKFEHCNFNDYTTRLR
ENEL
DIPP
(146)
Glycosylation of
printed peptides for
peptide diversification
for mapping of
glycopeptide specific
abs
GalNAc-T2 and
T4
MALDI-TOF/MS
Peptides derived from
M. musculus podoplanin
DIPP
(147)
Histone Deacetylases (HDACs), Sirtuins (Sirts), ADP-Ribosyl-transferases (ARTs)
Detection of ADPribosylation sites
pertussis toxin (B.
pertussis)
32P
NAD+ and
phosphorimaging
5- to 25-mer C-terminal
peptides of the GProtein -subunits of
Gi3, Gi, Gs, Go1, Go2,
GoX1, Trod, Gz, Gq/11 and
Gh, partial substitutional
analysis of the 16-mer
C-terminal peptide of the
Gi3 -subunit
SPOT
(148)
Determination of
substrate specificity
Pea HDAC
complexes HD1
and HD2 (P.
sativum)
Free amino groups
generated by
HDAC action were
chemically
acetylated,
detection of 14Clabelled acetyl
residues by
autoradiography
Overlapping peptides
containing KAc from
histones H3 and H4
SPOT
(149)
Analysis of the
influence of local and
distal substrate
HDAC8
MALDI-TOF/MS
(SAMDI assay)
Peptides derived from
the first 19 aa of H4:
truncated peptides, R
DIPP
(150)
substitutions, KAc and
methylated variants,
deletion and extensions
variants and
substitutions
sequences on activity
Assay for HDAC
profiling
HDAC1-3, HDAC8
and HDAC3
togehter with
SMRT
MALDI-TOF/MS
(SAMDI assay)
361 Member acetylated
peptide library:
GXKAcZGC, X, Z ≠ Cys
DIPP
(151)
Detection of ADPribosylation sites
SIR2 (T. brucei)
32P
NAD+ and
phosphorimaging
Overlapping peptide
scan of B. taurus H1.1
DIPP
(152)
Substrate specificity
determination and
identification of novel
in vivo substrates
Human Sirt3
Anti-Sirt3 ab
followed by HRPconjugated
secondary anti-IgG
ab
229 Peptides derived
from known acetylation
sites and 300 lysinecentered 9meric
peptides randomly
selected from
mitochondrial proteome
SPOT
(153)
Substrate specificity
determination and
identification of isoform
specific substrates
HDAC8, Sirt1,
HDAC2,
HDAC3/SMRT,
Hela Jurkat and
smooth muscle
cell lysate and
nuclear Hela
lysates treated ±
thymidine and
nocodazole
MALDI-TOF/MS
(SAMDI assay)
361 Peptides
GRKAcXZC, X, Z ≠ Cys
and 54 peptides to
investigate the function
of the Arg residue Nterminally to KAc
DIPP
(154)
Identification of in vivo
substrates
Human Sirt3
Anti-Sirt3 ab
followed by HRPconjugated
secondary anti-IgG
ab
240 peptides derived
from known
mitochondrial acetylation
sites
SPOT
(155)
Substrate specificity
determination
Chymotrypsin,
papain
Fluorescence of
released FITClabelled peptide
fragment
400 dipeptides
Identification of key
residues in protease
inhibitors
Pancreatic
elastase (S.
scrofa) and third
domain of turkey
ovomucoid
inhibitor
(OMTKY3)
Chemoluminescenc
e mediated by POD
fused to protease
Substitutional scan of 3
OMTKY3 derived
peptides, substitutional
analysis and length scan
Proteases
SPOT
SPOT
(156)
(157)
Substrate specificity
determination
Outer membrane
protease T
(OmpT) (E.coli),
trypsin
Fluorescence
increase by
clavage of
internally queched
Dap(Dnp)/Abz
peptides
Library screen: AGXA,
P2RRA, AP1RA, ARP-1A,
ARRP-2
Identification of
protease inhibitors
Cathepsin C,
cathepsin L
Fluorescence
imaging of
fluorescein labelled
PNA-peptide
conjugates
Putative Cys protease
inhibitors fused to PNA
derivatives
Identification of
proteases responsible
for transferrin receptor
cleavage
Purified proteases
A80 and A85
(cathepsin G,
neutrophil
elastase) ± anti
neutrophil
elastase ab (AHN10) and inhibitors
prefabloc SC,
aprotinin,
elastatinal, E64,
phosphoramidon,
pepstatin A,
EDTA, DMSO
Protease SPOT
assay –
Fluorescence of
released Abzlabelled peptide
fragment
19 Transferrin receptor
derived peptides and
substitutions
Assay for monitoring of
proteolytic activity
Caspase 3, Jurkat
cell lysate ±
granzyme B and
inhibitors
Fluorescence
imaging of
fluorescein labelled
PNA-peptide
conjugates
7 Peptidic acrylates as
protease inhibitors fused
to PNA derivatives
Substrate cleavage
site determination
Serine protease
DegP (E.coli)
Protease SPOT
assay –
Fluorescence of
released Abzlabelled peptide
fragment
Overlapping peptide
scans of PapA
Determination of
substrate specificity
Trypsin,
gramzyme B,
thrombin
Fluorescence
increase of
deacylated aminocoumarine
derivative
Ac-Leu-Gly-Pro-LysACC-linker, Ac-Nle-ThrPro-Lys-ACC-linker, and
Ac-Ile-Glu-Pro-AspACC-linker or 361member library of
general structure Ac-AlaXaa1-Xaa2-Lys-ACClinker with Xaa1=Xaa2=
all proteinogenic amino
acids except cysteine)
SPOT
DCSD
SPOT
DCSD
SPOT
DIPP
(158)
(159)
(160)
(161)
(162)
(163)
Protease assay using
microarrays containing
glycerol nanodroplets
and aerosol deposited
substances
Thrombin,
chymotrypsin and
caspase 2 ±
corresponding
CHO-inhibitors,
thrombin ±
benzamidine,
plasminogen
activator,
urokinase, factor
Xa, plasmin,
kallikrein and
caspases 2, 4, 6
for compound
library incubation
± VEID-MCA or
LEHD-MCA
Fluorescence of
released methoxycoumarylamine
Boc-VPR-MCA and 2
BODIPY labelled casein
substrates, peptide-MCA
substrates: VEID, LEHD,
YVAD, DEVD, VDVAD,
IETD and 352
compound library
Method for solutionbased screening of
protease substrate
specificity
Caspase 3,
thrombin, plasmin
and apoptotic and
non apoptotic cell
lysate
Fluorescence
imaging,
rhodamine-labelled
protease substrate
linked to PNA,
NleTRP, DEVD and 192
member split and mix
tetrapeptide library:
R1R2R3R4, R1 = D, R, L,
R2 = F, V, T, P, R3 = D,
R, T, P, R4 = D, R, P,
Nle, spatial
deconvolution by
oligopeptide microarray
hybridization
Protease activity
measurement using
semi-wet
supramolecular
hydrogel
Lysyl
endopeptidase,
chymotrypsin, V8
protease and
inhibitors
Fluorescence
increase of
released DANSyl
derivative
SSSSK-linker-DANSyl,
SSSSE-linker-DANSyl,
DANSyl-FKSSKS
Determination of
substrate requirements
and development of a
hK2 avtivated prodrug
for prostate cancer
therapy
Glandular
kallikrein 2 (hK2),
trypsin
Protease SPOT
assay –
Fluorescence of
released Abzlabelled peptide
fragment
Semenogelin I and II
derived peptides with Nterm. Abz, for the best
two substrates aa
substitutions in P1’ and
Lys or His substitutions
in P1 and fluorescence
quenched combinatorial
library with radom
sequences in P3-P1:
YIGKAXXX, X ≠ Cys
Method using real-time
SPR imaging to obtain
multiplexed kinetic
informations and
analysis of the
sequence specificity of
Factor Xa
real-time SPR
imaging (anti-Flag
ab)
Prothrombin factor Xa
recognition sequences
CSGIEGRDYKDDDDK,
CSGIEGADYKDDDDK,
CSGDYKDDDDK
SPNS
DCSD
Diff
SPOT
DIPP
(164)
(165)
(166)
(167)
(168)
factor Xa
Substrate specificity
determination
APC, kallikrein,
factor VIIa and TF,
IXa, XIa, XIIa,
complement C1r,
C1s, complement
factor D, trypsin,
tryptase, subtilisin
carlsberg,
cathepsin B, G, H,
K, L, S, V,
rhodesain, papain,
chymopapain,
ficain, stem
bromelain
Fluorescence
increase of
released aminocoumarin derivative
722 Member library:
P4P3P2P1, P4 = Ala, P3,
P2 ≠ Cys, P1 = Lys, Arg
Substrate specificity
determination
Thrombin,
plasmin, factor Xa,
uPA, thrombin (B.
taurus, S.
salmothymus)
Fluorescence
increase of
released aminocoumarin derivative
722 Member library:
P4P3P2P1, P4 = Ala, P3,
P2 ≠ Cys, P1 = Lys, Arg
Identification of
optimized substrates
for blood proteases
Kallikrein, factor
Xa, thrombin,
uPA, plasmin and
plasma treated ±
Ca2+, tissue
factor, kaolin, uPA
and 10 plasma
samples and 10
proteases
(kallikrein, factor
Xa, XIIa, XIa, IXa,
VIIa, thrombin,
APC, uPA,
plasmin)
Fluorescence of
released methoxycoumarylamine
Z-FR-MCA, Boc-IEGRMCA, Boc-VPR-MCA,
glutaryl-GR-MCA, BocVLK-MCA and 361
compound library: AcXXXX-ACC
Identification of
substrates
ADAM8
Protease SPOT
assay –
Fluorescence of
released Abzlabelled peptide
fragment
34 Peptides derived
from proteins involved in
inflammatory processes
and immune response in
the nervous system and
various peptide derived
from MBP cleavage sites
Analysis of protease
cleavage specificity
Chymopapain,
subtilisin
Fluorescence
increase by
clavage of
internally queched
peptides(FAM/TAM
RA)
10000 Member PNA
encoded library:
TAMRA-ßAla-Ser-Xaa4Xaa3-Xaa2-Xaa1-AlaLys(FAM)-TtdsLys(PNA)-amide with
TAMRA =
Quencher/Normalization
SPNS
SPNS
SPNS
SPOT
DCSD
(169)
(170)
(171)
(172)
(173)
fluorophor; FAM =
Fluorophor, Ttds = PEGbased-linker moiety, Xaa
= random positions with
amino acid residues Ala,
Asp, Phe, Lys, Leu, Asn,
Pro, Ser, Val, Tyr.
Monitoring caspase
activity
Purified caspase 3
or lysates of CHO
cells treated with
staurosporin
Fluorescence
imaging
subsequent to
protease treatment
ADEVDA and negative
control AEVEEA flanked
by Cy3 fluorophore and
QSY7-quencher moiety.
Positive control without
quencher
Assay for measuring
caspase activities in
cell lysates
Caspase 3 and 8,
SKW6.4 and
Jurkat (caspase 8
or deficient) lysate
treated ±
LzCD95L or
staurosporine
MALDI-TOF/MS
(SAMDI assay)
DEDVAFC, IETDAFC,
CGGDEVDSG,
CGDEVDSGVDEVA,
CGKRKGDEVDSG,
CGGIETDSG,
CDGIETDSG,
CDGIETDSGVDDD,
CGELDSGIETDSG
Surface optimization
for SPOT synthesis
Chymotrypsin
fluorescence
imaging
subsequent to
treatment with Cy5labelled
streptavidin
Biotinylated peptides of
general structure biotinGA-P1-G-linker with P1=
A,F, G, I, L, P, V, W
SPR based assay for
monitoring protease
activities
Caspase 3, 6, 8,
CHO lysate
incubated ±
staurosporine and
inhibitor DEVDCHO
Surface plasmon
resonance imaging
subsequent to
treatment with
protease followed
by streptavidin
Caspase substrates
DEVD, VEID, IETD and
negative controls EVEE,
VIEE, TIEE,
respectively, flanked by
cysteinyl-PEG-linker Nterminally and by
GGSK(biotinyl)-amide Cterminally
Multiplexed solutionphase assay for
protease profiling
Trypsin,
chymotrypsin,
endoproteinases
Asp-N, Glu-C,
Lys-C, Arg-C,
thrombin, factor
Xa, HRVI,
caspases, MMPs,
enterokinases,
tobacco etch virus
protease and
Jurkat cell lysate
incubated ± Fas
Protease cleavage
separates His-tag
from biotin residue
allowing removal of
uncleaved peptides
by streptavidin
beads, readout
using anti-His-tag
ab
Over 1000 DNA
encoded, biotinylated
peptide derivatives with
pentaHis-tag
representing substrates
for several protease
groups
DIPP
DIPP
SPOT
DIPP
DCSD
(174)
(175)
(176)
(177)
(178)
ab or Bcl2
overexpressing
Jurkat lysate
Design of biosensors
Botulinium
neurotoxin and
light chain of
botulinium
neurotoxin
FRET activity
assay yielding
fluorescence
increase
subsequent to
proteolytic
cleavage
SNAP-25 peptide
substrate
(FITC/DABCYL labelled)
Substrate specificity
determination
Subtilase 3 S.
lycopersicum
Protease SPOT
assay –
Fluorescence of
released Abzlabelled peptide
fragment
72 Peptides derived
from systemin and
substitution analysis
using A, F, P, S, N, D, K,
V, L, and H for P5 to P3´position
SPOT
(180)
Validation of protease
assay
Caspase 3,
caspase 7,
chymotrypsin,
subtilisin,
thrombin, trypsin
Fluorescence
increase of
deacylated
rhodamine
derivative
10 Peptide sequences
derived from known
substrates C-terminally
linked to fluorophore
DIPP
(181)
Substrate specificity
comparison
Granzyme A,
granzyme K and
inactive variants
Fluorescence
decrease
subsequent to
treatment with
fluorescein labelled
streptavidin
1000 Biotinylated
random peptides and 24
controls
DIPP
(182)
Monitoring of
proteolytic activities
onto fluorous based
microarrays
Thrombin,
plasmin,
chymotrypsin,
trypsin, granzyme
B
Fluorescence
increase of
deacylated
aminocoumarine
derivative
Bz-FVR-ACC, Bz-VPRACC, Ac-IEPD-ACC,
Cbz-FR-ACC, Suc-AFKACC; Suc-AAPF-ACC,
Bz-FVR-AMC, Bz-VPRAMC, AC-IEPD-AMC,
Cbz-FR-AMC, Suc-AFKAMC, Suc-AAPF-AMC
DIPP
(183)
Assay for hydrolytic
enzymes (proteases,
esterases, epoxid
hydrolases,
phosphatases)
Epoxide hydrolase
(R. rhodochrous),
acetylcholin
esterase (E.
electricus), trypsin
(B. taurus), AP (B.
taurus)
Enzyme recognition
head coupled to
fluorogenic moiety
which serves as a
reporter group that
translates
enzymatic activity
into fluorescence
readouts
Asp and Lys derivatives
DIPP
(184)
Novel based assay for
identification of
HRP, AP, -Gal
Fluorescently
labelled substrate
10000 Random peptide
DIPP
(185)
Diff
(179)
Miscellaneous
modulators of enzyme
function
analoga
sequences
CLPP = Cross Linking of Pre-synthesized Peptides
DCSD = DNA /PNA Chips as Sorting Device
Diff. = Different technologies
DIPP = Directed Immobilization of Pre-synthesized Peptides
DPCC = Deposition of Peptide-Cellulose Conjugates (Celluspots)
LDPS = Light Directed Peptide Synthesis
SODA = Synthesis On Defined Areas
SPNS = Solution Phase and Nano Spray
SPOT = SPOT Synthesis of Peptides
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