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

Summary

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.

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 enzymes^1-3. 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 reactions^10-11. 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

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; acid-catalyzed carboxyl oxygen exchange reaction) and B) analysis by mass spectrometry and graphical representation of ¹⁸O-incorporation ....

Results

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.......

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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