This work was supported by NIH/NIDCR Grant 1R01DE019796.

The presented LeO-workflows allow for the stable isotope labeling of protein digests and synthetic peptides. These time course experiments (Figure 1) are applicable to comparative and quantitative proteomics studies as well as protease research. Each workflow consists of two experimental steps (Figure 2): A) The time resolved sampling of the respective 18O-stable isotope-encoded reaction (protease-catalyzed peptide cleavage; protease-catalyzed carboxyl oxygen exchange reaction...

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No conflicts of interest declared.

By combining stable isotope labeling and high-resolution mass spectrometry in a time-resolved manner, the PALeO-TimeCourse method allows for a dynamic analysis of the generation of peptide products. The assay can be used to generate stable isotopically labeled peptides for quantitative and qualitative proteomics studies and to evaluate the kinetics by which proteotypic peptides are generated. Furthermore, PALeO-TimeCourse is designed to evaluate proteolytic pathways under specific, physiologically relevant conditions <em...

<table border="1">
 <tbody>
 <tr>
 <td>Name of material</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>PepClean C-18 Spin Columns</td>
 <td>Thermo</td>
 <td>89870</td>
 </tr>
 <tr>
 <td>Opti-TOF 384 MALDI target plate </td>
 <td>AB SCIEX </td>
 <td>1016629</td>
 </tr>
 <tr>
 <td>4800 MALDI TOF/TOF</td>
 <td>AB SCIEX </td>
 <td></td>
 </tr>
 </tbody>
</table>
Table 1. Materials
<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Alpha cyano-4-hydroxycinnamic acid</td>
 <td>Sigma Aldrich</td>
 <td>70990-1G-F</td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA)</td>
 <td>Sigma Aldrich</td>
 <td>A3294-10G</td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>Acros</td>
 <td>16568-0050</td>
 </tr>
 <tr>
 <td>Iodoacetamide (IAM)</td>
 <td>Sigma Aldrich</td>
 <td>1149-5G</td>
 </tr>
 <tr>
 <td>Endothelin converting enzyme-1 (ECE-1)</td>
 <td>R&amp;D Systems</td>
 <td>1784-ZN</td>
 </tr>
 <tr>
 <td>Trypsin Gold</td>
 <td>Promega</td>
 <td>V5280</td>
 </tr>
 <tr>
 <td>Water-18O, 97 atom % 18O</td>
 <td>Sigma Aldrich</td>
 <td>329878-1G</td>
 </tr>
 <tr>
 <td>Trifluoroacetic acid (TFA)</td>
 <td>Thermo</td>
 <td>28904</td>
 </tr>
 <tr>
 <td>Mass Standards Kit for Calibration of AB SCIEX TOF/TOF instruments</td>
 <td>AB SCIEX</td>
 <td>4333604</td>
 </tr>
 </tbody>
</table>
Table 2. Reagents

protease- and acid-catalyzed labeling workflows employing 18o-enriched water

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.

We used the PALeO-TimeCourse workflow to dynamically monitor the incorporation of 18O-stable isotopes into peptide cleavage products generated by proteolytic enzymes. The presented approach is a versatile tool to comparatively study proteolytic processing pathways for different substrate and protease combinations. By sampling proteolytic reactions repeatedly over the course of the reaction, the PALeO-TimeCourse experiment provides time-resolved snapshots of substrate and product abundances and processing detai...

Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

protease-acid-catalyzed-labeling-workflows-employing-18o-enriched

Research

JoVE Journal

Biology

11.9K visualizações.  Boston Biomedical Research Institute. Fluxos de trabalho de isótopos estáveis ​​de rotulagem empregando 18 O-água enriquecida (Leo-fluxos de trabalho) são ferramentas versáteis para estudos quantitativos e qualitativos proteômica. Nos fluxos de trabalho protease assistidas (Paleo), 18 O-átomos são introduzidos por clivagem proteolítica e reacções de permuta de oxigénio carboxilo mediada por proteases. No catalisada por ácido de fluxo de trabalho (Aleo),