Cross-linking mass spectrometry (XL-MS) is a structural biology technique that can provide insights into the structure and interactions of proteins and their complexes, especially those that cannot be easily studied by other methods. Quantitative XL-MS has the potential to probe the structural and temporal dynamics of protein complexes; however, it still requires further development. Until recently, quantitative XL-MS has largely relied upon isotopic labeling and data dependent acquisition (DDA) methods, which limits the number of comparable systems that can be studied in a single experiment. Here, the acquisition modes available on an ion mobility separation (IMS) enabled oa-ToF mass spectrometer are evaluated for the quantitation of cross-linked peptides, eliminating the need for isotopic labels and thus expanding the number of comparable studies that can be conducted in parallel. Workflows were optimized using metabolite and peptide systems, facilitating modelling of the data and saturation correction issues, which allow for small but significant increase in dynamic range. Evaluation of the DDA acquisition method, most commonly-used in XL-MS studies, indicated consistency issues between technical replicates and relatively poor performance in quantitative metrics. On the contrary, data independent acquisition (DIA) and parallel reaction monitoring (PRM) modes proved more robust in analyte quantitation, with the former being adapted using concepts of a Hi(n) approach to determine the molar amounts of cross-linked peptides relative to their linear counterparts. Mobility enabled acquisition methods exhibited varied results, whereby the added dimension of separation improved signal to noise and sensitivity, but also resulted in a simultaneous reduction in dynamic range, which was largely recovered by two correction methods.

Liquid chromatography. Reversed-phase gradient separations were conducted with I class or M-class liquid chromatography (LC) systems (Waters Corporation) for the quantitative metabolite and peptide experiments, respectively. For metabolite analysis, an I class system was equipped with an HSS T3 1.8 µm 2.1 x 100 mm column (Waters Corporation) operated at 400 µL/min. The gradient was held at 1% B for 0.3 min, followed by a 1 to 50% increase in from 0.3 to 7 min, and another step from 50 to 70% B in 1 min. Next, the solvent strength was increased to 99% B in 0.1 min, which was held for 1 min, and the column reconditioned for 1 min at initial gradient conditions. Mobile phase A was 0.1% formic acid in water and mobile phase B 0.1% formic acid in acetonitrile. The column temperature was maintained at 45 °C and the samples at 8 °C. The injection volume equaled 5 µL. All peptide separations were conducted with a 1.7 µm CSH 130 C18 300 µm x 100 mm column (Waters Corporation) operated at 7 µL/min using an M-class system. Here, the gradient was held first at 1% B for 2 min, followed by a 1 to 30% increase from 2 to 30 min, which was held for 2 min. Next, the solvent strength was increased to 85% B in 1 min, which was held for another 2 min, followed by decreasing the solvent strength in 1 min and reconditioning of the column for 23 min at initial gradient concentration. Mobile phase A was 0.1% formic acid in water and mobile phase B 0.1% formic acid in acetonitrile. The column temperature was maintained at 55 °C and the samples at 12 °C. The injection volume equaled 4.5 µL. Mass spectrometry. All MS experiments were conducted with an IM enabled Synapt G2-Si hybrid quadrupole oa-ToF mass spectrometer (Waters Corporation, Wilmslow, UK). For the quantitative metabolite analysis experiments, the capillary voltage was 1.0 kV, sampling cone 25 V, source offset 30 V, source temperature 100 °C, desolvation temperature 600 °C, cone gas 50 L/hr, desolvation gas 1000 L/hr, and nebulizer pressure 6 bar. Similar conditions were used for the quantitative peptide and cross-linked peptide analysis experiments, where the capillary voltage was 2.3 kV, sampling cone 30 V, desolvation temperature 250 °C, desolvation gas 500 L/hr, and nebulizer pressure 6 bar. The source offset and source temperature were not changed, and cone gas disabled. For the IM enabled acquisition methods, the Trap and Transfer T-Waves were pressurized with 2 mL/min of Ar. Gas-phase optimization for the separation of the analytes made both use of N2. The He gate contained within the mobility region was pressurized with 180 mL/min. The IM T-Wave was pressurized with 90 mL/min, the ion mobility wave velocity was ramped and the pulse height held at 40 V during acquisition.

Eight metabolite standards were supplied at a concentration of 100 ng/µL methanol. These included AKB-48 Apinaca 5 Hydroxypentyl metabolite, AKB-48 Apinaca 5 Hydroxy-pentyl metabolite-D4, JWH-073 3 Hydroxybutyl metabolite, JWH-073 3-Hydroxybutyl metabolite-D5 (indole-D5), JWH-250 4-Hydroxypentyl metabolite, JWH-250 4-Hydroxypentyl metabolite-D5, JWH-122 4-Hydroxypentyl metabolite, and JWH-122 4 Hydroxypentyl metabolite-D5 (Sigma-Aldrich, St. Louis, MO). Four further standard solutions were obtained by diluting the initial stock of each metabolite into human urine, prepared as described elsewhere and liquid chromatography mass spectrometry (LC-MS) QC Reference Mixture (Waters Corporation, Milford, MA).19 The final metabolite concentrations equaled 1 ppb/µL, 10 ppb/µL, 100 ppb/µL, and 1000 ppb/µL urine, respectively. MassPREP Protein Digestion Standard Mix 1 (Waters Corporation), consisting of tryptic digested Yeast Enolase, Glycogen Phosphorylase B, Yeast Alcohol Dehydrogenase and Bovine Serum Albumin (BSA), was spiked a constant background of 100 ng/µL of MassPREP E. coli digest standard (Water Corporation), as well as LC-MS QC Reference Standard. Five further standard solutions at concentrations of 10 amol/µL, 100 amol/µL, 1 fmol/µL, 10 fmol/µL, and 100 fmol/µL E. coli solution were obtained by serial dilution of a 1 pmol/µL Protein Digestion Standard Mix 1 stock solution. Cross-linking reactions were conducted as described previously.20 In short, 0.3 mg/mL BSA (Sigma-Aldrich) and 1 mg bis(sulfosuccinimidyl)suberate (BS3) d0/d12 (Creative Molecule Incorporated) were prepared in 20 mM HEPES at pH 7.6. The cross-linker was added to the protein and diluted to a final concentration of 2.5 mM BS3 d0/d12. The sample was then incubated at room temperature for 40 min under mild agitation. Following incubation, the reaction was quenched by adding 1 M NH4HCO3 to a final concentration of 50 mM. The samples were then evaporated to dryness and re-suspended in 8 M urea at 1.1 mg/ml concentration. 1% RapiGest (Waters Corporation) was added to a final concentration of 0.1 and incubated with 10 mM dithiothreitol (DTT) at 37 °C for 30 min. Following incubation, the sample was cooled to room temperature. Iodoacetamine (IAA) was added to a final concentration of 20 mM and the sample incubated in the dark at room temperature for 30 min. The sample was then diluted with 50 mM NH4HCO3 to reduce the final concentration of urea to < 1 M. Trypsin, 50:1 protein to enzyme (w/w), was added to the sample and the reaction incubated overnight at 37 °C with mild agitation. Following overnight incubation, enzymatic activity was quenched by adding formic acid to a final concentration of 2% (v/v). The sample was purified using Sep-Pak SPE cartridges (Waters Corporation) and evaporated to dryness. The cross-linked bovine serum albumin (XL-BSA) sample was reconstituted and spiked at different concentrations into a constant background of 100 ng/µL MassPREP E. coli Digest Standard to provide standards, based on original BSA concentration, of 1 amol/µL, 10 amol/µL, 100 amol/µL, 1 fmol/µL, 10 fmol/µL, and 100 fmol/µL E. coli solution.