Protease और एसिड उत्प्रेरित लेबलिंग रोजगार वर्कफ़्लोज़<sup18></sup> हे समृद्ध जल
Summary
स्थिर आइसोटोप लेबलिंग workflows रोजगार<sup18></sup> O समृद्ध पानी (LEO workflows) मात्रात्मक और गुणात्मक प्रोटिओमिक्स अध्ययन के लिए बहुमुखी उपकरण हैं. Protease की सहायता (Paleo) workflows में,<sup18></sup> O-परमाणुओं proteolytic दरार और carboxyl ऑक्सीजन विनिमय प्रतिक्रियाओं proteases से मध्यस्थता से शुरू कर रहे हैं. कार्यप्रवाह एसिड उत्प्रेरित (Aleo) में,<sup18></sup> O-परमाणुओं कम पीएच में carboxyl ऑक्सीजन विनिमय से शुरू कर रहे हैं.
Abstract
Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such “double-labeling” enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.
Protocol
Representative Results
Discussion
स्थिर आइसोटोप लेबलिंग और एक समय हल तरीके से उच्च संकल्प मास स्पेक्ट्रोमेट्री के संयोजन, paleo – TimeCourse विधि पेप्टाइड उत्पादों की पीढ़ी के एक गतिशील विश्लेषण के लिए अनुमति देता है. परख मात्रात्मक और गुणात्मक …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
यह काम NIH / NIDCR 1R01DE019796 अनुदान द्वारा समर्थित किया गया.
Materials
| Name of material | Company | Catalogue number |
| PepClean C-18 Spin Columns | Thermo | 89870 |
| Opti-TOF 384 MALDI target plate | AB SCIEX | 1016629 |
| 4800 MALDI TOF/TOF | AB SCIEX |
Table 1. Materials
| Name of reagent | Company | Catalogue number |
| Alpha cyano-4-hydroxycinnamic acid | Sigma Aldrich | 70990-1G-F |
| Bovine serum albumin (BSA) | Sigma Aldrich | A3294-10G |
| Dithiothreitol (DTT) | Acros | 16568-0050 |
| Iodoacetamide (IAM) | Sigma Aldrich | 1149-5G |
| Endothelin converting enzyme-1 (ECE-1) | R&D Systems | 1784-ZN |
| Trypsin Gold | Promega | V5280 |
| Water-18O, 97 atom % 18O | Sigma Aldrich | 329878-1G |
| Trifluoroacetic acid (TFA) | Thermo | 28904 |
| Mass Standards Kit for Calibration of AB SCIEX TOF/TOF instruments | AB SCIEX | 4333604 |
Table 2. Reagents
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