Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery

Abstract

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Activity-based Protein Profiling Approaches for Transplantation

“…The labeled proteins were affinity purified as previously described. 19,42 Following affinity purification, there was an increase in the number and intensity of the labeled proteins with a marked reduction in the total protein content in the affinity-purified samples ( Figure 4B). The in-gel patterns of the probe-labeled proteins in the starting materials were similar for subclinical and clinical rejection urines, with increased activities in the purified materials, and this type of result provides evidence for enrichment of the activity probe-labeled enzymes ( Figure 4C).…”

“…These observations were consistent with early intraoperative enzyme activity changes that preceded protein transcription/translation. 19 Enzymes are proteins that catalyze the chemical reactions required for physiological function. A review of the Uniprot protein database indicates that >3400 entries are associated with enzymatic activity, highlighting the importance of this category of proteins.…”

“…47 Urines were reacted with a fluorescent-tagged serine hydrolase activity probe (fluorophosphonate-TAMRA, FP-TAMRA), and probe-labeled samples were separated by SDS-PAGE using previously described methods. 19,42 An illustrative example of the serine hydrolase activities in normal transplant, subclinical, and clinical rejection urines is shown ( Figure 3A). This example demonstrates differences in the activity bands between the 3 patient groups in complexity and intensity and the presence of interindividual variability within each group.…”

“…Indeed, enzyme activities may not directly correlate with enzyme quantity, emphasizing the critical importance of characterizing protein function. 19,42 The illustrative example highlights 2 areas of activity that do not correlate with total protein levels in the corresponding regions of the gel (Figure 3B). This suggests that serine hydrolase enzymes detected by the activity probe may be low-abundance proteins and not necessarily the major proteins in the corresponding gel region.…”

“…Ultimately, the affinity-purified materials can be identified using mass spectrometry. 19,42 As the patterns of probe reactivity may vary depending on the probe structure, it is beneficial to use probes with overlapping target enzyme repertoires to maximize coverage for in-depth enzyme identification. An example of the serine hydrolase activities that are common and unique to the FP-TAMRA and 6-N-biotinylaminohexyl isopropyl phosphorofluoridate (PF-biotin) activity probes in the same patient sample pool is illustrated in Figure 4A.…”