Rojas Echeverri JC, Milkovska-Stamenova S, Hoffmann R. A Workflow towards the Reproducible Identification and Quantitation of Protein Carbonylation Sites in Human Plasma. Antioxidants (Basel). 2021 Mar 1;10(3):369. doi: 10.3390/antiox10030369. PMID: 33804523; PMCID: PMC7999155.
Keywords:
protein carbonylation; human plasma; aldehyde reactive probe (ARP); biotin-avidin affinity; LC-MS/MS
Submitter: Juan Camilo Rojas Echeverri
Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their repro-ducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to RPC ESI-MS/MS-TWIMS. The presented workflow addresses several analytical challenges by us-ing ARP specific fragment ions to reliably identify ARP-peptides. Furthermore, the reproducible recovery and relative quantitation of ARP-peptides was validated. HSA in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive car-bonyl species (RCS) with most RCS modifications were detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of HSA.
Covalently linked carbonyls from human plasma proteins and an oxidized human serum albumin protein model were derivatized with aldehyde reactive probe (ARP), a biotinylated, carbonyl-specific derivatization reagent. Proteins were digested with trypsin with a FASP procedure and peptide digest was processed with biotin-avidin chromatography. Both peptides in fractions prior to enrichment and in the elution fractions were analyzed with LC-TWIMS-MS/MS with a Q-IMS-TOF instrument (Synapt G2-Si) in DDA mode.
Schematic presentation of the analytical workflow applied to plasma samples for ARP-peptide enrichment (left) and the following LC-MS-based analysis from acquisition to data processing (right). Ultrafiltration of blood plasma was applied to simultaneously remove small molecules and reconstitute proteins in acidic conditions (I) to derivatize carbonylated proteins with ARP (II). Proteins were digested with trypsin using a FASP approach (III) and the resulting peptide mixture was split and either mixed with an ARP-labelled digest of a model protein (III.A) or directly enriched by avidin-affinity chromatography (IV). The fractions were submitted to ultrafiltration to remove interfering monomeric avidin (V) and analyzed by nRPC‑ESI‑MS/MS-TWIMS in DDA mode (VI). The generated tandem mass spectra were processed with a hybrid de novo and database search approach (VII) considering specific ARP fragmentation patterns. All proposed ARP-peptides were validated by manual annotation of the mass spectra and considering both drift times in IMS and retention times in RPC (VIII). The filtered peptide list and corresponding peak areas were further processed (IX) to assess recovery, sample preparation workflow reproducibility, and protein carbonyl modification site location.
Experimental setup used to evaluate the enrichment reproducibility (ARP-Plasma:ARP-OxHSA data set; panel a), the matrix interference effects (Plasma:ARP-OxHSA data set; panel b), and the reproducibility of the whole procedure (ARP-Plasma Upscale data set; panel c).
Panorama_sample_list.xlsx