Normal ICAT Samples labeled (2 hrs) and trypsin-digested (overnight) at 37°C

1. Un-Labeled Peptide (2Hr labeling) Un-Labeled Peptide (10 min labeling) Fig. 3: TIC of depleted rat serum, with 1:1 CEM-labeled light:heavy peptides. 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. Higher-throughput Cleavable ICAT Analysis using Microwave TechnologyTonya M. Pekar, Mai-Loan Nguyen, Jennifer Rutherford, David Innamorati, Joe Bonapace, and John PirroCharles River Proteomic Services, Worcester, MA Results: Introduction: 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. Full Range Standard 2 Hr Labeling and Cleaving Cleaved Laminin Un-cleaved Laminin 10 min CEM Labeling and Cleaving Cleaved Laminin Un-cleaved Laminin 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 CEM 1:1 Fig. 2: MALDI spectra of CEM-labeled and normal-labeled BSA; 1:1 light:heavy. Fig. 5: MS/MS spectrum of light-labeled peptide; fragments used to ID parent protein and identify label. • 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 cation-exchange, 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 • Normal ICAT • Samples labeled (2 hrs) and trypsin-digested (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 • 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 proteinID’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 increase low abundant protein ID’s.