Chemicals were purchased from common commercial sources. Pepsin from porcine gastric mucosa was from Sigma-Aldrich (Steinheim, Germany, lot no. SLBQ1670V), 2D-Quant Kit from GE Healthcare (Freiburg, Germany), NaOCl solution for decontamination from Carl Roth (Karlsruhe, Germany), deuterated atropine (d3-Atr) from CDN Isotopes (Pointe-Claire, Quebec, Canada), and hair shampoo from a common commercial provider. SM was provided by the German Ministry of Defense and checked in-house for integrity and purity by NMR. Working solutions of SM were prepared in acetonitrile yielding concentrations ranging from 15 µM to 50 mM. Scalp hair was a donation from 7 human non-exposed male and female individuals. Additional beard, abdominal, armpit, and pubic hair was obtained from one male individual. Pooled scalp hair was produced by mixing equal amounts of minced hair from all donors. Sweat was collected from the skin of several volunteers after intense physical activity and purified from particles by ultrafiltration (UF) prior to use (Amicon Ultra-0.5 centrifugal filter unit, 0.5 mL, molecular weight cut-off, MWCO, 10 kDa, Merck Millipore, Billerica, Massachusetts, USA).
Initial rinsing of hair
Hair was rinsed three times with methanol (70% v/v) and three times with H2O prior to air drying (16 h, room temperature, RT). Afterwards, the hair was cut into pieces (2–3 mm length) and stored at RT until use.
Incubation of hair with SM
Pieces of hair (10 mg in total) were covered with SM working solutions (150 µL) for references (25 mM SM) and standards (diverse SM concentrations) and with neat acetonitrile (150 µL) for blanks and mixed with H2O (450 µL, each). After 30 min, the mixture was centrifuged (15,000 RCF, 10 min, 25 °C), the liquid phase was discarded, and hair was washed three times with acetonitrile (200 µL). Subsequently, the hair was dried at RT for 1 h and stored at – 20 °C prior to use. Scalp hair from seven individuals and pooled hair as well as scalp, beard, abdominal, armpit, and pubic hair from one individual was incubated (n = 3, each).
Lysis of hair
Lysis of hair was performed based on the Shindai method (Fujii et al. 2013). In brief, following the standard protocol, lysis buffer (700 µL, 20 mM Tris–HCL, 2.6 M thiourea, 5 M urea, 20 mM dithiothreitol, pH 9.0) was added to hair (10 mg) and shaken constantly for 4 h (50 °C). After centrifugation (15,000 RCF, 10 min, 25 °C), 500 µL was transferred into a reaction vial followed by carbamidomethylation with iodoacetamide (26 µL, 1 mM) under gentle shaking in the dark (1 h, 50 °C). During method optimization, different periods of lysis were tested (0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h).
Proteolysis of keratins
Following the standard protocol, the reaction mixture obtained after lysis and carbamidomethylation (500 µL) was subjected to UF (15,000 RCF, 10 min, 35 °C). The retentate was washed 2-times by UF after the addition of H2O (300 µL) and subsequently two times after the addition of formic acid (FA, 300 µL, 10% v/v). Proteolysis of proteins in the retentate was performed by adding pepsin solution (100 µL, 2.5 mg/mL in FA 10% v/v) and FA (300 µL, 10% v/v) under constant shaking (2 h, 42 °C). During method optimization, diverse periods for proteolysis were tested (1.5 min, 3 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, 270 min, and 300 min). After UF (15,000 RCF, 15 min, 20 °C), 30 µL of the filtrate was mixed with 60 µL d3-Atr solution (3 ng/mL in FA 0.5% v/v) prior to µLC–ESI MS/HR MS analysis. Measurements were performed using either an Orbitrap system working in data-dependent tandem-MS mode (ddMS2) or a quadrupole time-of-flight (TOF) system operating in product ion scan (PIS) mode.
μLC-ESI MS/HR MS (ddMS2) analysis
For identification of alkylated peptides by µLC–ESI MS/HR MS, the ddMS2 mode was carried out using a QExactive plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) working with positive ESI. The common instrumental equipment and software used were the same as described recently (Blum et al. 2020; John et al. 2021). An Acquity HSS T3 column (50 × 1.0 mm I.D., 1.8 µm, 100 Å, Waters, Eschborn, Germany) protected by a precolumn (Security Guard™ Ultra Cartiges UHPLC C18 peptide 2.1 mm I.D., Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation of 20 µL sample volume at 30 °C with a linear gradient of solvent A (0.05% v/v FA) and solvent B (acetonitrile/H2O 80:20 v/v, 0.05% v/v FA) at a flow of 30 µL/min: t [min]/B [%]: 0/0; 3/0; 35/40; 35.5/95; 39.5/95; 40/0.
The ddMS2 approach was comprised of an initial full MS scan followed by MS/MS of the ten most intense ions (top 10) (John et al. 2021). The following parameters were applied for full MS: spray voltage 3.5 kV, capillary temperature 250 °C, sheath gas flow rate 23 arbitrary units (a.u.), mass spectrometric resolution at full width at half maximum (fwhm) 70,000; automatic gain control (AGC) target 3.0 × 106, maximum injection time (MIT) 100 ms, and scan range m/z 200—m/z 1500. For ddMS2, the following settings were used: fwhm 17,500, AGC target 1.0 × 105, MIT 150 ms, isolation window 1.6 m/z, normalized collision energy (NCE) 30; minimum AGC target 3.0 × 103, intensity threshold 2.0 × 104, charge inclusion 2–7, and dynamic exclusion 30 s.
MS/MS spectra of peptides fragmented by ddMS2 were analyzed by the Proteome Discoverer 2.1 software (Thermo Scientific) and measurements were matched to a database containing amino acid sequences of all hair keratins (K 31–K 40, K 81–K 86) taken from the UniProt database considering the SM-derived HETE-attachment (+ 104.030 Da at Cys, Asp, Glu, His, Met) and carbamidomethylation (+ 57.021 Da at Cys) using the Sequest HT algorithm. Peptides proposed to bear SM alkylation were manually checked for the presence of a signal at m/z 105.037 representing the cleaved [HETE]+-moiety. The identity of proposed alkylated peptides was subsequently confirmed by µLC–ESI MS/HR MS analysis in PIS mode.
μLC–ESI MS/HR MS (PIS) analysis
For µLC–ESI MS/HR MS (PIS) analysis, a microLC 200 pump (Eksigent Technologies LLC, Dublin, CA, USA) on-line coupled to a TripleTOF 5600+ mass spectrometer (TT5600+, ABSciex, Darmstadt, Germany) was used as described recently (Schmeißer et al. 2021). Chromatographic separation (30 °C, 30 µL/min) of 20 µL sample was performed on an Acquity HSS T3 (100 × 1.0 mm I.D., 1.8 µm, 100 Å, Waters), protected by a precolumn as described above. After 5 min of equilibration under starting conditions, a gradient of solvent A and solvent B was applied: t [min]/B [%]: 0/5; 4/29; 12.5/32; 16/50; 16.5/95; 18.5/95; 19/5; 20/5.
Product ions of the double-protonated alkylated peptides AE(-HETE)IRSDL, FKTIE(-HETE)EL, and LE(-HETE)TKLQF were obtained after collision-induced dissociation (CID) and monitored in the mass range from m/z 50 to m/z 950 with an accumulation time of 100 ms, each. Individual masses of precursor ions and of the two most intense product ions (qualifier I and qualifier II) are listed in Table 1. The following MS parameters were the same for the three alkylated peptides: collision energy spread (CES) 3 V, curtain gas (CUR) 2.07 × 105 Pa (30 psi), heater gas (GS1) 2.76 × 105 Pa (40 psi), turbo ion spray gas (GS2) 3.45 × 105 Pa (50 psi), ion release delay (IRD) 67 ms, ion release width (IRW) 25 ms, ion spray voltage floating (ISVF) 5000 V, and temperature (TEM) 200 °C.
Selectivity of the µLC–ESI MS/HR MS (PIS) method and interindividual differences in biomarker yield
Blank and reference hair from 7 donors (scalp, 10 mg, n = 3, each) as well as blank and reference scalp, beard, abdominal, armpit, and pubic hair (10 mg, n = 3, each) from one individual were prepared according to the standard protocol and analyzed by µLC–ESI MS/HR MS (PIS) to monitor any interference in blanks and to compare individual biomarker peak areas in references.
Limit of identification of biomarker peptides
Pooled scalp hair (10 mg) was incubated with SM working solutions (n = 3) to generate standards corresponding to SM concentrations of 12 µM, 24 µM, 48 µM, 96 µM, 190 µM, 390 µM, 780 µM, 1.56 mM, 3.125 mM, 6.25 mM, 12.5 mM, 25 mM, and 50 mM. Following the standard protocol, relative peak area ratios of the respective qualifier II to qualifier I of the three biomarker peptides (Table 1, given in percent) were calculated. The limit of identification (LOI) was defined as the lowest concentration of SM at which all measurements of the triplicate met the area ratios obtained from pooled hair reference.
Stability of biomarker peptides in the autosampler
To evaluate the stability of the three biomarker peptides stored in the autosampler at 15 °C, pooled hair reference (scalp, 10 mg) was prepared according to the standard protocol and analyzed by µLC–ESI MS/HR MS (PIS) hourly within a period of 24 h. Biomarker peak areas were determined to monitor relative concentration–time profiles.
Time-dependent adduct formation in the presence of water
Pooled scalp hair (10 mg) was mixed with SM working solution (150 µL, 25 mM) and additionally with either H2O or acetonitrile (450 µL, each). The liquid phase was discarded after 1 min, 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 60 min, 90 min, and 120 min (tested as separate incubation mixtures, n = 3, each). Immediately afterwards, acetonitrile (200 µL) was added to the hair for vortex-mixing. Removal of the liquid phase and addition of another acetonitrile portion (200 µL) was repeated five times. Following standard lysis and proteolysis, the samples were analyzed by µLC–ESI MS/HR MS (PIS) to monitor the relative time-dependent concentrations of all three biomarker peptides.
Influence of sweat on adduct formation
Pooled hair (scalp, 10 mg) was incubated with SM either in the presence of H2O (450 µL, n = 3) or in the presence of sweat (450 µL, n = 3). Subsequent sample preparation and analysis were done according to the standard protocol to determine the peak areas of the three biomarker peptides.
SDS-PAGE of lysed hair and in-gel proteolysis with pepsin
After lysis of blank and pooled hair reference (scalp, 10 mg, n = 3, each), the supernatant containing 30 µg protein (quantified by 2D-Quant Kit) was separated by SDS-PAGE (NuPAGE™ 4–12% bis Tris Gel, Thermo Fisher Scientific, Waltham, MA, USA). Subsequent staining with Coomassie Brilliant Blue (CBB) and in-gel proteolysis (2 h) were carried out according to common protocols (Thermo Fisher Scientific 2021) but using pepsin (2.5 mg/mL FA 10% v/v) instead of trypsin. The three biomarker peptides were detected by µLC–ESI MS/HR MS (PIS).
Stability of alkylated keratins in hair during storage
To characterize the stability of alkylated keratins in hair, pooled hair reference (scalp, 10 mg, n = 63) was stored under the following conditions: – 20 °C in the dark and exclusion of air (condition I, n = 21); 25 °C in the dark and exclusion of air (condition II, n = 21) and 25 °C with daylight and contact to air (condition III, n = 21). Hair was analyzed weekly (n = 3 per condition) according to the standard protocol over a period of 7 weeks monitoring the relative concentration–time profiles of all three biomarker peptides. In addition, the period of analysis for condition III was prolonged for another 7 week period (14 weeks in total).
Stability of alkylated keratins in hair during wash cycles with shampoo
Pooled hair reference (scalp, 10 mg) was mixed with 500 µL of a water–shampoo mixture (H2O/shampoo 4:1 v/v) and shaken for 5 min (40 °C). After centrifugation (15,000 RCF, 10 min, 25 °C), the supernatant was removed and the procedure was repeated five times with 500 µL H2O instead of the water–shampoo mixture. Finally, the hair was dried at RT for 24 h. This entire wash cycle with shampoo and water was repeated on 4 consecutive days. After each cycle, samples (n = 3) were processed according to the standard protocol and analyzed by µLC–ESI MS/HR MS (PIS). Peak areas of all biomarker peptides were compared to those of references not subjected to any wash cycle.
SM is a highly toxic blister agent. Only trained personnel wearing laboratory protective clothes should handle it under the fume hood. Decontamination of all materials that have come into contact with SM is mandatory. Decontamination should be done by submerging the material into alkaline NaOCl solution and leaving it in the solution for several hours.