Higher-throughput Cleavable ICAT Analysis using
Microwave Technology
Tonya M. Pekar, Mai-Loan Nguyen, Jennifer Rutherford, David Innamorati,
Joe Bonapace, and John Pirro
Charles River Proteomic Services, Worcester, MA
Introduction:
Results:
Full Range
Cleaved Laminin
The power of combining
cleavable ICAT (isotope-coded
affinity tags) and CEM
microwave technologies offers a
more efficient, high-throughput
analysis of differential
quantitation and identification in
both simple and complex protein
mixtures.
The time required to process
ICAT-labeling protocols has been
reduced from 10+ hours to 30
minutes. The technique has been
applied to both BSA for
validation and depleted rat serum.
Here we demonstrate the
successful labeling and
identification of 76 proteins from
depleted serum.
Methods:
Depletion
• Sprague dawley rat serum was depleted of
RSA-IgG-Transferrin
• ABI vision system
• Avian IgY antibodies
• UltralinkTM hydrazide beads
Microwave-ICAT
• CEM DiscoverO’ microwave system
• Samples were microwave-labeled with
light (C12) or heavy (C13) biotinylated
labels (10 min)
• Labeled fractions were mixed 1:1 and 1:2
• Samples were microwave-digested with
trypsin (10 min), cleaned via cationexchange, purified on an avidin cartridge,
and microwave-cleaved of biotin (10 min)
CEM DiscoverO’ Protocol
• 10 minute digest
• Max power: 50 W
• Max temp: 60ºC
• 10% cooling
Un-Labeled Peptide
(2Hr labeling)
Standard 2 Hr Labeling and Cleaving
Un-cleaved Laminin
Cleaved
Laminin
Un-Labeled Peptide
(10 min labeling)
Un-cleaved Laminin
10 min CEM Labeling and Cleaving
Fig. 1: MALDI spectra of normal and CEM-labeled laminin peptide.
Zoom
Normal 1:1
CEM 1:1
Fig. 4: MS spectrum of corresponding light and heavy
peptides.
Normal 1:1
Fig. 3: TIC of depleted rat serum, with 1:1 CEM-labeled
light:heavy peptides.
CEM 1:1
Fig. 2: MALDI spectra of CEM-labeled and normal-labeled
BSA; 1:1 light:heavy.
Normal ICAT
• Samples labeled (2 hrs) and trypsindigested (overnight) at 37°C
• Samples cleaned/purified as above, and
cleaved (2 hrs) at 37°C
Kratos Axima MALDI-TOF
• Digests C18 zip-tipped and eluted with
2mg/mL HCCA in 90%AcCN, 0.1% TFA
on hydrophobic probe
ABI Q-STAR/LC-MS/MS
• Digests seperated on a Swift C18 column
(150 µm i.d. x 5 cm, 5 µm particle size),
250 min separation--6-35% B
• Analyzed positive ion mode, 1 sec MS
scan, 3 sec MS/MS scan, 2.6 kV
ProICAT Analysis
• ABI ProICAT software was used for
analysis of the data dependent scans
Fig. 5: MS/MS spectrum of light-labeled peptide; fragments used to ID
parent protein and identify label.
Accession #
Name
Confidence Peptides Found Avg H:L
gi|125520
T-KININOGEN I PRECURSOR
99
31
0.8402
gi|1345834 C-REACTIVE PROTEIN PRECURSOR
99
3
0.9576
gi|130318
PLASMINOGEN PRECURSOR
99
2
0.9567
gi|2497917 ZINC-ALPHA-2-GLYCOPROTEIN PRECURSOR
99
1
1.0339
gi|1730193 UDP-GLUCOSE 4-EPIMERASE
95
1
0.7502
Fig. 6: Example of the protein identification and quantitation output of ProICAT software used for analysis of the data dependent scans. 76 protein identifications were
made from MS/MS of cysteine-labeled peptides, confidence >64.
Conclusions:
• The time required for cleavable-ICAT analysis has been reduced from 10+ hours to 30 minutes.
• The combination of depletion and microwave-assisted ICAT enhances low abundant protein ID’s.
• 76 proteins were identified with a confidence of >64 using ABI ProICAT software.
Future Work:
• Application of ABI iTRAQ labels to biological samples.
• Comparison of the relative efficiency of microwave-labeling to normal labeling.
• Increase the number of depleted proteins from serum/plasma samples and pre-fractionation prior to analysis to