Prolidase

LC-MS grade methanol and acetonitrile were obtained from Fisher Chemical (Loughborough, UK). Pepsin, pronase E, sulfuric acid and creatinine were from Merck (Darmstadt, Germany), and CML and [ 2 H 2 ]CML were obtained from PolyPeptide Group (Strasbourg, France). 2-Amino-2-(hydroxymethyl)propane-1, 1-diole-hydrochloride (TRIS-HCl) was purchased from Serva Feinbiochemica (Heidelberg, Germany). Nonafluoropentanoic acid (NFPA), hydrochloric acid, prolidase, leucine aminopeptidase and n-heptane were from Sigma-Aldrich (Seelze, Germany). Lithium citrate buffer, lithium citrate/borate buffer, lithium hydroxide and ninhydrin were purchased from Sykam (Fürstenfeldbruck, Germany). Wieninger's catalyst was obtained from Honeywell (Seelze, Germany). Boric acid was purchased from Carl Roth (Karlsruhe, Germany) and sodium hydroxide from Grüssing (Filsum, Germany). Water was distilled (twice) before analysis.
Pyrraline [11] and MG-H1 [12] were synthesized according to the specified literature. The synthesis of [ 13 C 6 , 15 N 2 ] pyrraline and [ 13 C 6 ]MG-H1 was performed in the same manner but using [ 13 C 6 , 15 N 2 ]lysine (pyrraline) and [ 13 C 6 ]arginine (MG-H1) instead of the unlabeled amino acids.

Bovine serum albumin, 2,4-dinitrophenylhydrazine (DNPH), l-methionine, pepsin (3839 U/mg protein), leucine aminopeptidase (18 U/mg protein), prolidase (553 U/mg protein) and pronase E (4,000 PU/mg) were purchased from Sigma-Aldrich (Steinheim, Germany). HPLC-MS/MS grade methanol and ethylene glycol were from Fisher Scientific (Loughborough, United Kingdom) and sodium dodecyl sulfate (SDS) and urea from Roth (Karlsruhe, Germany). Guanidinium hydrochloride, 5,5ʹ-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent, DTNB), and l-MetSO 2 were obtained from Alfa Aesar (Karlsruhe, Germany). Reduced glutathione and ninhydrin were purchased from Merck (Darmstadt, Germany) and l-MetSO from Bachem (Bubendorf, Switzerland). Tin(II) chloride from Laborchemie (Apolda, Germany) was used. The water used for the preparation of buffers and solutions was double-distilled (Destamat Bi 18E; QCS GmbH, Maintal, Germany). The [ 2 H 3 ] and [ 2 H 8 ] isotopologues of MetSO and MetSO 2 as well as pyrraline were synthesized as published previously [21, 22] .

Unless otherwise stated, chemicals were purchased from commercial suppliers without further purification: NaCl (≥ 99.5%, Fisher Scientific), NaOH (97%, VWR chemicals), HCl (≥ 37%, VWR chemicals), H 2 SO 4 (> 96%, VWR chemicals), heptafluorobutyric acid (HFBA, 99%, Alfa Aesar), nonafluoropentanoic acid (NFPA, 97%, Sigma Aldrich), pepsin (3839 U/mg protein, Sigma Aldrich), pronase E (4000 PU/mg, Merck), leucine aminopeptidase (18 U/mg protein, Sigma Aldrich), prolidase (553 U/mg protein, Sigma Aldrich), acetonitrile (HPLC-MS/MS grade, VWR), tris-(hydroxymethyl)-aminomethane (TRIS, > 99.9%, Carl Roth), bidistilled water, N-ε-benzoyllysine, CML, [ 2 H 2 ] CML and furosine (PolyPeptide). The following substances were synthesized according to methods stated in the literature: Pyrraline [23, (link)24] (link), MG-H1 [25] (link), CEL [25] (link), maltosine [26] (link), pentosidine [27] (link). The syntheses of [ 13 C 6 , 15 N 2 ] pyrraline, [ 13 C 6 ] MG-H1 and [ 13 C 6 , 15 N 2 ] maltosine were performed in the same manner but using [ 13 C 6 , 15 N 2 ] lysine (pyrraline and maltosine) and [ 13 C 6 ] arginine (MG-H1) instead of the unlabeled amino acids. For the synthesis of [ 13 C 3 ] CEL, [ 13 C 3 ] pyruvic acid was used instead of unlabeled pyruvic acid.