Development of an immobilized-trypsin reactor coupled to liquid chromatography and tandem mass spectrometry for the analysis of human hemoglobin adducts with sulfur mustard
Introduction
As a strongly alkylating agent, sulfur mustard (HD) can react with biological nucleophiles such as proteins to form hydroxyethylthioethyl (HETE) adducts, that can be used as unequivocal biomarkers of exposure after accidental or intentional poisoning to the chemical agent [1]. Particularly useful biomarkers are the adducts that HDforms with hemoglobin, one of the most abundant proteins found in human blood (average concentration of 140 mg⋅mL−1) [2], which can be detected up to several months after an exposure to this chemical warfare agent (CWA). Indeed, these long-lived adducts have a life-time similar to that of the native protein, i.e. 120 days [3].
As a complement to the modified Edman degradation procedure, mainly used to analyze the adducts that HD forms with the N-terminal valine residues of both globin α and globin β chains constituting hemoglobin [4], [5], [6], a new analytical method was recently developed in our laboratory based on the LC-MS/MS analysis of the alkylated peptides resulting from the tryptic digestion of hemoglobin incubated in vitro with HD [7]. This method allowed the confirmation of 10 adduction sites among the 11 sites previously identified in the literature (α-Val1, α-His20, α-His45, α-His50, β-Val1, β-Glu22, β-Glu26, β-Glu43, β-His77 and β-His97) [8], [9], but also led to the identification of 5 new alkylation sites (α-His72, α-His87, α-His89, β-His2 and β-Val98). The localization of the different adduction sites is illustrated on the Fig. 1. Moreover, this method appeared to be repeatable (RSD ranging from 0.5 to 9.3%) and sensitive, with the possibility to detect less than two adducted chains over one million, and a satisfying linearity was observed for all 15 adducted peptides targeted on the HD concentration range studied (250 ng⋅mL−1 – 100 µg⋅mL−1). However, this method was applied to the analysis of alkylated peptides resulting from the in-solution digestion of hemoglobin, which is time-consuming. Indeed, due to the auto-digestion of the protease, in-solution digestion must be performed with low Enzyme/Substrate (E/S) ratios, thus leading to long digestion times and the digestions are often carried out overnight (16–18 h). Moreover, the enzyme cannot be reused for other digestions which can lead to high costs depending on the protease used. Furthermore, since in-solution digestions are carried out off-line, the automatization of this step is rather complicated. Immobilized enzyme reactors (IMERs) have several advantages compared to in-solution digestion. First of all, due to the immobilization of the protease, the auto-digestion of the enzyme is highly reduced [10]. Therefore, the digestions can be performed with higher E/S ratios, which leads to digestion times shorter than in solution. Finally, the IMERs are reusable, which reduces the cost of the analysis on a long term, and the digestion is easily automated and can be coupled on line with the LC-MS/MS analysis of peptides. The first study related to the use of an IMER for proteins analysis goes back to the late 1980s [11]. In this study, an IMER based on trypsin immobilized on an agarose gel and conditioned in a Pyrex tube was used for the characterization of proteins at low concentrations. Since then, many studies based on IMERs prepared with different grafting supports and conditioned in different formats have been reported in the proteomics filed. Indeed, the solid phase can be conditioned in multiple formats such as cartridges, disks, pre-columns and pipette tips [12]. Recently, using a pepsin IMER conditioned in a 30 × 2.1 mm column, this approach has already proven its potential and its efficiency to detect adducts formed by organophosphorus nerve agents with human butyrylcholinesterase (HuBuChE) in plasma [13]. Several reviews present the studies carried out with IMERs for proteins analysis and highlight the advantages of the digestion on these supports, especially the reduction of the digestion time from several hours to few minutes while obtaining sequence coverages similar to those resulting from in-solution digestion [14], [15], [16], [17], [18], [19], [20].
Therefore, to improve the performance of the analysis of hemoglobin adducts and overcome the limitations of in-solution digestion, the development of an IMER for the digestion of adducted hemoglobin was proposed. For this, the selected enzyme for the proteolysis was trypsin, due to the high specificity of its cleavages that makes it possible to easily compare the experimentally obtained peptides with the theoretical ones. While commercial IMERs are available, such as the commercial Poroszyme® device composed of trypsin immobilized on a cross-linked co-polymer, previous studies have showed that laboratory-made IMERs led to a more efficient digestion [21], [22]. Moreover, enzyme grafting yields on commercial devices are often not available while the laboratory-made IMERs present the advantage of being able to control the amount of enzyme immobilized on a chosen support, but also to select the reactor dimensions in order to develop a quality on-line set-up with the analytical column. Finally, using a grafting support such as Sepharose leads to very low non-specific interactions. This study is the first one, to our knowledge, to introduce the use of trypsin-based reactors for the digestion of human hemoglobin exposed to HD.
After controlling the repeatability of the grafting of trypsin on Sepharose, some parameters that may affect the digestion of the protein on IMER were studied using non-alkylated hemoglobin. This study allowed settling the temperature, the digestion duration and the volume required to transfer the protein from the injection loop to the trypsin IMER. These digestion conditions were applied to the digestion of hemoglobin on three independently synthetized trypsin IMERs, to evaluate the inter-IMER and intra-IMER digestion repeatability, and the results were compared to those resulting from in-solution digestions. The on-line digestion on IMER was then applied to the analysis of hemoglobin in vitro exposed to different concentrations of HD in pure water, and the results were compared with those obtained after off-line in-solution digestion of the same samples. Particularly, the linearity, sensitivity and repeatability of both procedures were compared.
Section snippets
Chemicals
KCl, KH2PO4 and acetonitrile (ACN) were purchased from VWR (Fontenay-sous-bois, France). NaCl, sodium hydrogen phosphate Na2HPO4, NaHCO3, NaCH3CO2, sodium azide (NaN3), calcium chloride dihydrate (CaCl2·2H2O) and Trizma® hydrochloride (NH2C(CH2OH)3.HCl, Tris-HCl) were provided by Sigma Aldrich (Saint Quentin Fallavier, France), as well as trypsin from bovine pancreas treated with 6-(1-tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK), hydrochloric acid (HCl) and formic acid. A Milli-Q water
Preparation of the trypsin IMERs and evaluation of their grafting yields
Three trypsin-based IMERs, namely IMER 1, IMER 2 and IMER 3, were synthetized on different days by immobilizing the enzyme on CNBr activated Sepharose following the procedure described in the Section 2.3. This agarose-based sorbent was chosen based on a previous study carried out in our laboratory [21] as it develops very low non-specific interactions and presents a good chemical stability. Grafting yields of 30%, 27% and 32% were respectively obtained for IMER 1, IMER 2 and IMER 3, E in good
Conclusion
After demonstrating the repeatability of the IMERs grafting process, it was also demonstrated that the digestion of hemoglobin on the IMERs can be achieved in 30 min and it was more repeatable than the conventional in-solution digestion achieved in 16 h. Moreover, the digestion is carried out directly on-line with the LC-MS/MS analysis of the resulting peptides, which could make the automatization of the whole procedure possible. Finally, this study proved that the three IMERs were similar in
Compliance with ethical standards
This is not a clinical study on humans/animals.
Funding
This work was supported by DGA, CBRN Defence [grant number 201870152].
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank the French Defense Procurement Agency (DGA) for its financial support and for providing alkylated hemoglobin solutions for the study.
References (23)
- et al.
Improvements in monitoring the N-terminal valine adduct in human globin after exposure to sulfur mustard and synthesis of reference chemicals
Talanta.
(2011) - et al.
Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of tryptic digest of human hemoglobin exposed to sulfur mustard
J. Chromatogr. B.
(2021) - et al.
Enzymatic microreactors in chemical analysis and kinetic studies
Biotechnol. Adv.
(2006) - et al.
Application of immobilized enzyme reactor in on-line high performance liquid chromatography: A review
J. Chromatogr. B.
(2005) - et al.
Immobilized enzyme reactors in proteomics, TrAC
Trends Anal. Chem.
(2011) - et al.
Proteolytic enzyme-immobilization techniques for MS-based protein analysis, TrAC
Trends Anal. Chem.
(2009) - et al.
Microscale immobilized enzyme reactors in proteomics: Latest developments
J. Chromatogr. A.
(2014) - et al.
Evaluation of various immobilized enzymatic microreactors coupled on-line with liquid chromatography and mass spectrometry detection for quantitative analysis of cytochrome c
J. Chromatogr. A.
(2008) - et al.
Development of immobilized-pepsin microreactors coupled to nano liquid chromatography and tandem mass spectrometry for the quantitative analysis of human butyrylcholinesterase
J. Chromatogr. A.
(2016) - et al.
Measurement of Protein Using Bicinchoninic Acid’
Anal. Biochem.
(1985)
Analysis for plasma protein biomarkers following an accidental human exposure to sulfur mustard
J. Anal. Toxicol.
Cited by (5)
Analysis of long-lived sulfur mustard-human hemoglobin adducts in blood samples by red blood cells lysis and on-line coupling of digestion on an immobilized-trypsin reactor with liquid chromatography-tandem mass spectrometry
2022, Journal of Chromatography ACitation Excerpt :Amicon® Ultra-4 mL (100 kDa and 50 kDa) and Centrisart® (100 kDa) centrifugal ultrafiltration devices were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). Regarding trypsin immobilization, the grafting solution and washing solutions were prepared as described in our previous study [16]. Non-spiked human blood sample (28 mL, batch 10,200,088,369) from a healthy individual without known exposure to sulfur mustard was provided by the center de Transfusion Sanguine des Armées (CTSA, Clamart, France).
Analytical methods based on liquid chromatography for the analysis of albumin adducts involved in retrospective biomonitoring of exposure to mustard agents
2024, Analytical and Bioanalytical ChemistrySample transformation in online separations: how chemical conversion advances analytical technology
2023, Chemical CommunicationsApplication Strategy of Mass Spectrometry in Protein Biomarker Discovery
2022, Chinese Journal of Modern Applied Pharmacy