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.
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.
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
II. PALeO-TimeCourse: Postdigestion labeling of proteolytic termini
III. ALeO-TimeCourse: Acid-catalyzed labeling of carboxyl groups
B. MALDI-TOF/TOF MS/MS Data Acquisition and Analysis
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).
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 details. Co-spotting samples with acidic matrix solution on a target plate stops the enzymatic reaction and matrix crystallization further preserves sample composition. Therefore, time points can retrospectively be analyzed by MALDI-TOF/TOF MS/MS after conclusion of the enzymatic reaction. To accommodate the data-rich nature of these experimental workflows, we implemented a semi-automatic bioinformatics system that calculates 18O-incorporation ratios for each peptide. Figure 3 shows a representative spectral time course plot of the processing of peptide Endokinin C by ECE-1. PALeO-TimeCourse data is multidimensional: The expanded view of the MS data across the entire mass range simultaneously shows the abundances of the peptide substrate as well as the abundances of the peptide products. The temporal arrangement allows deciphering the sequence of cleavage events and determining which peptide fragments are stable cleavage products. The zoomed-in view of the MS data reveals the isotope envelopes of each peptide and cleavage-induced 18O-labeling is readily identified by its characteristic isotope distribution. 18O-labeling allows for positive selection of cleavage products for subsequent identification by MS/MS. Altogether, the PALeO-TimeCourse assay captures the dynamics of proteolytic processing and allows evaluating preferred cleavage sites of proteases. Yet another level of information can be obtained depending on the enzymatic mechanism of the utilized protease. Certain proteases such as the serine protease trypsin are capable of covalently rebinding their peptide cleavage products. The hydrolysis of the acyl-enzyme intermediate results in the incorporation of a second 18O-atom in the C-terminal carboxyl group. Figure 4 shows the resulting shifts in the relative contribution of the different isotopomers over time: The initial peptide bond cleavage reaction results in a 1:1 split between the 16O and 18O1 peptide fractions. The carboxyl oxygen exchange reaction results in the increase of the 18O2 fraction to the 25% ratio at equilibrium with a concomitant decrease of the 16O fraction. Proteases capable of catalyzing the carboxyl oxygen exchange reaction can therefore be used for an additional 18O-labeling workflow, the postdigestion labeling of proteolytic termini. In these types of experiments, substrates are initially digested in the absence of H218O, peptide products cleaned up and incubated with a fresh batch of protease, but this time in the presence of 50% H218O. Figure 4 shows the differences in isotope incorporation that can be observed in these two experimental workflows and the differences in incorporation speed between individual peptide substrates. In the postdigestion labeling workflow, the rate for both 18O-incorporations is determined by the carboxyl oxygen exchange reaction. The reaction rate depends on how readily the protease interacts with its cleavage products. The affinity for the reaction product may indirectly be used to infer the affinity of the protease to the original peptide substrate. Such information could be useful to determine which peptide sequences are ideal substrates for example for tryptic digests in quantitative proteomics studies. Peptides that are readily cleaved and double-labeled by trypsin are likely to be proteotypic peptides that are reproducibly and quantitatively formed and therefore ideally suited for comparative proteomics studies.
At low pH, oxygens of carboxylic acid groups exchange with the aqueous solvent19-20. This reaction can be used as an alternative approach to protease-catalyzed labeling strategies to introduce stable isotopes into peptides. In the ALeO-TimeCourse (acid-catalyzed labeling employing 18O-enriched water) workflow, we monitored the slow incorporation of 18O-atoms into synthetic peptides. The acid-catalyzed oxygen exchange stopped after co-crystallization with the matrix solution on the MALDI target plate, effectively “freezing” the isotope distribution state. We used Angiotensin 1 as a model peptide and examined its isotope envelope over time (Figure 5). An uptake of two 18O-atoms for each carboxyl group was observed. Under the current experimental conditions, the acid-catalyzed carboxyl oxygen exchange reaction was much slower than the protease-catalyzed exchange9 and reached equilibrium only after 48 days. However, the ALeO approach offers the advantage of labeling peptides that are not recognized by proteases. In addition, peptides incorporate multiple 18O-atoms depending on the number of acidic side chains, which can result in full separation of the isotope envelopes of unlabeled and labeled species. The overall mass shift provides information on the number of acidic residues in a given peptide and their location can be derived by the analysis of MS/MS fragment ion mass shifts. 18O-labeled peptides display near identical chromatographic behavior as their unlabeled counterparts, which enables their comparative analysis at identical elution time. In summary, acid-catalyzed 18O-labeling can serve as an alternative to chemical, enzymatic and metabolic labeling approaches commonly used in quantitative proteomics. One particular promising application of this technique is the use of 18O-labeled peptides as stable isotope standards.
Figure 1. 18O-based labeling offers a variety of time course workflows. (I) The protease-catalyzed PALeO-TimeCourse workflow allows for the monitoring of the dynamics of proteolytic cleavage reactions. Depending on protease, these reactions result in (a) single (e.g. in the case of certain metalloproteases) or (b) double 18O-incorporation (e.g. in the case of certain serine proteases). (II) Double 18O-labeler such as trypsin can also be used in postdigestion labeling workflows, in which 18O-incorporation is solely mediated by the protease-catalyzed carboxyl oxygen exchange reaction. (III) In contrast, the ALeO-TimeCourse workflow relies on the acid-catalyzed carboxyl oxygen exchange reactions of acidic peptide side and terminal groups. Click here to view larger figure.
Figure 2. Experimental workflows of the PALeO- and ALeO-TimeCourse strategies. A) (I) In a protease-catalyzed PALeO-TimeCourse experiment, substrates are incubated with a protease in the presence of H218O resulting in the incorporation of up to two 18O-atoms depending on the protease. (II) In a PALeO postdigestion labeling experiment, cleavage products from a previous digestion are re-incubated with a protease of interest in the presence of H218O. Proteases capable of double labeling (in workflow I) will catalyze the incorporation of two 18O-atoms. (III) In an acid-catalyzed ALeO experiment, all acidic functional groups incorporate 18O-atoms. B) TimeCourse Analysis: At timed intervals, aliquots of the reaction mixtures are co-spotted with matrix on MALDI-target plates. Upon MS-data acquisition, spectral time plots of the peptide cleavage reactions as well as 18O-incorporation plots of individual peptide species are generated and cleavage products are selected for MS/MS-based sequence identification. Click here to view larger figure.
Figure 3. Spectral time plots display the dynamics of proteolytic cleavage reactions. By plotting MS spectra of PALeO-TimeCourse experiments in a waterfall arrangement, it is possible to simultaneously monitor the degradation of the substrate and the emergence of intermediate and final cleavage products. Here, the cleavage of the bioactive peptide Endokinin C by Endothelin-converting enzyme-1 (ECE-1) is shown. Cleavage products were identified by MS/MS and their isotope envelopes displayed the characteristic 18O-incorporation signatures (highlighted in red).
Figure 4. Serine proteases such as trypsin mediate the incorporation of up to two 18O-atoms into peptide cleavage products. (I) In the protease-based labeling workflow, the peptide bond cleavage reaction results in a 50% single 18O-incorporation ratio for freshly generated peptide cleavage products (black dots indicate unlabeled, red single-labeled peptide fractions). Proteases that rebind their reaction products (e.g. serine proteases such as trypsin) further catalyze the incorporation of a second 18O atom via the carboxyl oxygen exchange reaction (green dots, double-labeled peptide fraction). At equilibrium, a label distribution of 0.25:0.5:0.25 (un-; single-; double-labeled) is reached. (II) In the postdigestion labeling of proteolytic termini workflow, no peptide bond cleavages occur. Instead, the incorporation of up to two 18O-atoms is exclusively based on the carboxyl oxygen exchange reaction. Therefore, 18O-incorporation only occurs with proteases that rebind their peptide cleavage products. Click here to view larger figure.
Figure 5. Acid-catalyzed 18O-labeling leads to the incorporation of two 18O-atoms per carboxyl group. Over the course of the experiment, the isotopic envelope of Angiotensin-1 underwent multiple +2 Da mass shifts (dashed lines) corresponding to the uptake of 18O-atoms. The sites of 18O-incorporation are highlighted in the amino acid sequence (blue).
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 ex vivo. Endogenous proteins, peptides and proteases as well as synthetic peptides and recombinant proteases can be utilized in the workflow. Depending on the particular proteolytic reaction to be investigated, assay conditions and spotting times can be adjusted to provide for optimal sampling of the reaction process. In our experience, it is helpful to slow proteolytic reactions down (e.g. by using low enzyme concentrations, room temperature) to allow for convenient manual sampling intervals. Like with other stable-isotope labeling methodologies, deviations for the experimental 18O-incorporation ratio measurements are small – typically less than 5% for peaks with sufficient ion statistics. The overall protocol can be easily adapted to other assay formats, for example the screening of a large set of synthetic substrates or the characterization of the proteolytic processing of endogenous peptides from complex biological samples (e.g., cell culture media, body fluids). We previously demonstrated how the PALeO-approach can be hyphenated with an LC-separation step, and used to define the dynamic composition of the human salivary peptidome9. Proteolytic signatures identified by the PALeO-TimeCourse provide important facts regarding the substrate specificity of the protease of interest. In addition, the time-resolved data also yields kinetic information. Such knowledge is particularly useful in the evaluation of proteotypic peptides for quantitative proteomics studies, where the choice of reproducible and highly abundant target peptides is essential21. Likewise, identifying rate-limiting steps in proteolytic pathways is of high interest for the development of novel protease-based drugs. Proteases are key factors in many physiological and pathological processes (e.g. cardiovascular disorders, neurodegenerative diseases, cancer) and such observations can open up new opportunities for therapeutic interventions. The ALeO-strategy – acid-catalyzed labeling of carboxyl groups – is easily applied to synthetic and endogenous peptides to produce stable isotopically encoded standards. The kinetics of the acid-catalyzed reaction is much slower than the enzyme-catalyzed reaction. Therefore, experiments have to be carefully planned ahead. ALeO-TimeCourse is a low-cost alternative to chemical, metabolic and synthetic labeling methods currently used in proteomics studies. The labeling process can be monitored to validate that 18O-incorporation reached equilibrium, which can be an advantage over other stable isotope labeling methods. In conclusion, the LeO-TimeCourse workflows described here are versatile tools that can creatively be employed in many qualitative and quantitative proteomics studies as well as protease research.
The authors have nothing to disclose.
This work was supported by NIH/NIDCR Grant 1R01DE019796.
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