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 kinetics.

A. TimeCourse Experiments

I. PALeO-TimeCourse: Protease-catalyzed labeling of proteolytic cleavages

1. (Optional) Disulfide bonds of proteins (10 μM) and peptides (250 μM) are reduced with DTT (final concentration 2.5 mM) in 25 mM NH₄HCO₃ (both freshly prepared) by incubation for 30 min at 50 °C.
2. (Optional) Free cysteines are alkylated with iodoacetamide (final concentration 10 mM) in 25 mM NH₄HCO₃ by incubation for 30 min at room temperature in the dark.
3. Depending on protease of interest, protein/peptide solutions need to be cleaned-up to remove residual buffer and alkylation agent. Use PepClean C-18 spin columns (Thermo) for peptide cleanup and Vivaspin Centrifugal Concentrators (Sartorius) to exchange buffers for protein samples.
4. Redissolve/exchange peptides/proteins in 20 μl protease reaction buffer (ECE-1: 50 mM MES-KOH, pH 5.5; trypsin: 25 mM NH₄HCO₃, pH 8.0) containing 1:1 (v/v) final H₂¹⁸O (95%, Sigma Isotec).
5. Withdraw a zero time point sample prior to the addition of the protease: Mix 0.5 μl of the reaction mixture and 0.5 μl alpha cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 50% acetonitrile, 0.1% TFA). Spot sample on an Opti-TOF 384 MALDI target plate (AB SCIEX) and leave the solvent droplets at room temperature until dry (about 5 min).
6. Split the reaction solution into two aliquots. The first aliquot will be incubated with the protease of interest (ECE-1: 75 nM; trypsin: 0.04 nM) at room temperature or the recommended temperature for the particular enzyme. The second aliquot will be incubated without protease and will serve as control sample. The first aliquot represents a standard sample for protease-catalyzed ¹⁸O-labeling and can be used for peptide and protein identification, detection of proteolytic activities and monitoring of proteolytic cleavage reactions. The second aliquot is used to show that ¹⁸O-incorporation is induced by the protease; therefore, neither labeling nor cleavage is expected for this sample.
7. Follow the reaction by removing reaction aliquots and spotting them as described under step 5 at time intervals as seem fit. The reaction conditions described above are used to monitor a proteolytic reaction for up to four days (approx. 24 spots). Start sampling every 5 min until 30 min, then spot every 30 min until 2 hr, then every hr until 8 hr and every 8 hr until 4 days. Cleavage products should appear within the first 12 hr and the substrate should be completely hydrolyzed after 24 hr. Extended reaction incubation times allow to define stable reaction products that are discrete from reaction intermediates, which are further processed. Depending on research question and given enzyme-substrate pairs, spotting times and incubation temperature have to be modulated to assure optimal reaction sampling.
8. After the final reaction time point is spotted or in between extended spotting intervals, the MALDI target plate is submitted to MALDI-TOF/TOF MS/MS analysis as described below (Section B).

II. PALeO-TimeCourse: Postdigestion labeling of proteolytic termini

1. (Optional) Disulfide bonds of proteins (10 μM) and peptides (250 μM) are reduced with DTT (final concentration 2.5 mM) in 25 mM NH₄HCO₃ (both freshly prepared) by incubation for 30 min at 50 °C.
2. (Optional) Free cysteines are alkylated with iodoacetamide (final concentration 10 mM) in 25 mM NH₄HCO₃ by incubation for 30 min at room temperature in the dark.
3. Depending on the protease of interest, protein/peptide solutions need to be cleaned-up to remove residual buffer and alkylation agent. Use PepClean C-18 spin columns for peptide cleanup and Vivaspin Centrifugal Concentrators for protein buffer exchange.
4. Redissolve/exchange the cleaned-up peptide products/proteins in 20 μl protease reaction buffer (e.g. trypsin: 25 mM NH₄HCO₃, pH 8.0) and digest with protease of interest to completion (e.g. trypsin: 0.04 nM; 37 °C; 12 hr).
5. Cleanup cleavage products with PepClean C-18 spin columns. This step will eliminate residual protease activities.
6. Redissolve/exchange the cleaned-up peptide products in 20 μl protease reaction buffer (trypsin: 25 mM NH₄HCO₃, pH 8.0) containing 1:1 (v/v) final H₂¹⁸O.
7. Withdraw a zero time point sample prior to the addition of enzyme: Mix 0.5 μl of the reaction mixture and 0.5 μl alpha cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 50% acetonitrile, 0.1% TFA). Spot sample on a MALDI target plate and leave the solvent droplets at room temperature until dry (about 5 min).
8. Split the reaction solution into two aliquots. The first aliquot will be incubated with protease of interest (e.g. trypsin: 0.04 nM) at room temperature or the recommended temperature for the particular enzyme. The second aliquot will be incubated without protease and will serve as a control. The first aliquot represents a standard sample for protease-catalyzed ¹⁸O-postdigestion labeling and can be used for peptide and protein quantification. The second aliquot is used to show that ¹⁸O-incorporation is catalyzed by the protease; therefore, no labeling is expected for this sample.
9. Follow the reaction by removing reaction aliquots and spotting them as described under step 7.) at time intervals as seem fit. For example, we spotted initially every 5 min for up to 30 min and every 15 min after that to monitor the carboxyl oxygen exchange reaction catalyzed by trypsin.
10. After the final reaction time point is spotted or in between extended spotting intervals the MALDI target plate is submitted to MALDI-TOF/TOF MS/MS analysis as described below (Section B).

III. ALeO-TimeCourse: Acid-catalyzed labeling of carboxyl groups

1. Incubate individual peptides (50 nM) with 1:1 (v/v) ¹⁸O-enriched water in the presence or absence (control) of 0.1% (v/v) final trifluoroacetic acid (total volume 30 μl).
2. Sample the reaction products daily for 48 days by co-spotting a 0.5 μl aliquot of the mixture with 0.5μl of alpha cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 50% acetonitrile, 0.1% TFA) directly onto a MALDI target plate.
3. Between spotting intervals and after spotting of the final reaction time point submit MALDI target plate for MALDI-TOF/TOF MS/MS analysis as described below in Section B.

B. MALDI-TOF/TOF MS/MS Data Acquisition and Analysis

1. Mass spectra are acquired on a 4800 MALDI TOF/TOF Analyzer (AB SCIEX).
2. Prior to analysis, the instrument is calibrated with a mixture of peptide standards (Mass Standards Kit for Calibration of AB SCIEX TOF/TOF instruments) with a maximum mass measurement error tolerance of ± 50 ppm and a minimum number of six peaks to match.
3. MS spectra (mass range 400 - 4,000 m/z) are acquired in triplicate using positive ion mode with an adjustable laser intensity (3,400 - 3,800; step size 50) with an acceptable base peak intensity range of 2,000 - 45,000. Single shots are acquired for sub-spectra, with 400 total shots/spectrum, stop conditions come into effect after 800 sub-spectra are acquired (pass or fail) or 400 sub-spectra pass acceptance criteria. In case of low-abundant samples or in the presence of complex biological backgrounds the lower end of the MS detection range should be raised to 800 m/z. In addition, it may be necessary to remove salt and other interfering compounds with a sample cleanup step as described earlier or by LC separation.
4. MS data files (.t2d files) are exported from the 4000 Series Explorer data acquisition software and imported into our in-house laboratory information system, which utilizes MASCOT Distiller software (Matrix Science) for spectral processing and peak detection. Isotopic envelopes are deconvoluted and ¹⁸O-incorporation ratios automatically determined using an algorithm similar to the one described by Mason et al.¹² and adapted by our group⁹. Alternatively, software tools such as ZoomQuant¹³ and Viper¹⁴, as well as commercial software packages such as BioWorks Xpress (Thermo Fisher Scientific) and Mascot Distiller Quantitation Toolbox (Matrix Science) can deconvolute ¹⁸O-type data^15,16. ¹⁸O-incorporation ratios are expressed as the relative contributions of individual peptide isotope species (i.e., peptides containing ¹⁶O, ¹⁸O₁ or ¹⁸O₂) to the entire isotopic envelope.
5. For each TimeCourse experiment, the molecular masses ([M+H]⁺) for all detected peptide species are extracted from the associated MS data files and the values binned at a 100 ppm mass width.
6. At least three [M+H]⁺ are set to be required to populate a bin. In case of known substrates, the filtered bin list is compared to a list of proteolytic cleavage products predicted from the substrate peptide sequence using the ExPASy FindPept tool¹⁷ (http://au.expasy.org/tools/findpept.html) and a 200 ppm mass error acceptance tolerance.
7. MS/MS spectra are acquired for all mass values of cleavage products predicted by the FindPept tool and for bin values that have ¹⁸O-incorporations associated with them. MS/MS data are acquired on the 4800 MALDI TOF/TOF Analyzer in 1kV reflector positive ion mode, with a fixed laser intensity of 4200 and CID-gas in off mode. 50 shots are acquired in a randomized pattern per sub-spectra up to a total of 40 sub-spectra per spot (yielding a total of 2,000 shots/spot).
8. MS/MS data files (.t2d files) are exported from the 4000 Series Explorer data acquisition software and imported into our in-house laboratory information system, peaks are detected and MS/MS peak lists are associated with the corresponding binned MS data.
9. For peptide identification, MS/MS peak lists are searched against the SwissProt database using the MASCOT search engine with the following search parameters: no enzyme specificity, 150 ppm precursor ion and 0.2 Da fragment ion mass tolerances.
10. Peptide identifications can additionally be validated using the Data Explorer software (AB SCIEX) by confirming the characteristic ¹⁸O-incorporation patterns across y-series fragment ions as described by Shevchenko et al.¹⁸.

C. Preparation of Spectral Time and ¹⁸O-incorporation Plots

Spectral time plots: MS data files (.t2d files) for each reaction time point are exported from the Data Explorer software as ASCII-files using a macro and imported into a data analysis and graphic software program (e.g. Origin by OriginLab) and displayed as waterfall plots (Figure 3).

¹⁸O-incorporation plots: For each binned peptide cleavage product, the relative contributions of individual peptide isotope species (¹⁶O, ¹⁸O₁ or ¹⁸O₂) are extracted across all reaction time points and plotted against time (Figure 4).

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