An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease

ARTICLE An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease Brad A. Palanski1,13,14, Nielson Weng1,2,3,14, Lichao Zhang 4, Andrew J. Hilmer1, Lalla A. Fall5, Kavya Swaminathan 6, Bana Jabri7,8,9, Carolina Sousa 10, Nielsen Q. Fernandez-Becker11, Chaitan Khosla1,5,12,15✉& Joshua E. Elias 4,15✉ Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes. https://doi.org/10.1038/s41467-022-28353-1 OPEN 1Department of Chemistry, Stanford University, Stanford, CA, USA. 2School of Medicine, Stanford University, Stanford, CA, USA. 3Medical Scientist Training Program, Stanford University, Stanford, CA, USA. 4Chan Zuckerberg Biohub, San Francisco, CA, USA. 5Stanford ChEM-H, Stanford University, Stanford, CA, USA. 6Division of Blood and Bone Marrow Transplantation, Stanford University, Stanford, CA, USA. 7Department of Medicine, University of Chicago, Chicago, IL, USA. 8Committee on Immunology, University of Chicago, Chicago, IL, USA. 9Department of Pathology and Pediatrics, University of Chicago, Chicago, IL, USA. 10 Facultad de Farmacia, Departamento de Microbiología y Parasitología, Universidad de Sevilla, Sevilla, Spain. 11 Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University, Stanford, CA, USA. 12 Department of Chemical Engineering, Stanford University, Stanford, CA, USA. 13 Present address: Department of Medicine, Brigham and Women’s Hospital, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. 14 These authors contributed equally: Brad A. Palanski, Nielson Weng. 15 These authors jointly supervised this work: Chaitan Khosla, Joshua E. Elias. ✉email: [email protected];[email protected] NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications 1 1234567890():,; In humans, the prevailing understanding of physiological digestion is that proteins are broken down into single amino acids or dior tri-peptides before absorption as nutrients1,2. Gluten proteins found in wheat, barley, and rye are exceptions to this tenet, which results in notable health consequences. Unusual biochemical properties of these proteins, such as a high abundance of glutamine (Gln, Q) and proline (Pro, P) residues, render them resistant to degradation by gastrointestinal proteases3. Consequently, relatively long gluten peptides with intact immunotoxic epitopes accumulate in the lumen of the small intestine4 and cross the epithelial barrier, although the mechanism(s) of transport remain controversial5. In approximately 1 in 100 individuals, an aberrant immune response to these peptides causes celiac disease (CeD), an autoimmune disorder that causes small intestinal mucosal injury characterized by villous atrophy. Common symptoms include abdominal pain, bloating, nausea, vomiting, and/or diarrhea6. Extraintestinal CeD manifestations also occur, including blistering skin rashes and ataxia7. At present, the only effective CeD treatment is a strict, lifelong adherence to a gluten-free diet (GFD)8. Assays to probe gluten immunogenicity in CeD have typically relied on extracted or recombinant glutens that are digested in vitro with gastrointestinal proteases. The resulting peptides can be used in bioassays to characterize mechanisms of immunotoxicity3. In most cases, these peptides elicit adaptive immune responses mediated by HLA-DQ2 or -DQ8 antigen presentation to Th1 cells in the small intestinal mucosa of patients with CeD4,9–14. Gluten peptides with alternative (i.e., non-T cell-dependent) modes of action have also been reported15–21. Structure-function analyses using synthetic gluten peptides have revealed exquisite sequence specificity for both the HLA and T-cell receptor; single amino acid alterations can result in dramatically altered affinities, thereby altering the strength of the immune response22–24. Although it is well established that chemical variation in gluten peptides can influence CeD immune responses, measuring their naturally processed forms and connecting them with CeD patients’health status is an underexplored research area. Over 20 years ago, chromatographic analysis coupled to UV detection implied the existence of gluten-derived peptides in the urine of patients with CeD25. This was confirmed more recently by antibody-based methods26–29. Indeed, most current gluten detection methods rely on monoclonal antibodies, which recognize amino acid motifs present in a subset of gluten proteins30. Notwithstanding the valuable knowledge that has been gained from analyzing biospecimens with these immunoreagents, they are neither capable of revealing the exact gluten peptide sequences that are present, nor are they comprehensive in that some CeD-relevant peptides may lack the epitopes these antibodies recognize. To address mounting evidence that many disease-relevant gluten peptides remain to be discovered31, new methods are needed to recover and precisely characterize in vivo gluten digestion products in an untargeted fashion. In fact, not a single chemically defined peptide from wheat (or, to our knowledge, from any dietary protein) has ever been identified from the human circulatory or excretory systems. Consequently, very little is known about the physiological absorption, distribution, metabolism, and excretion (ADME) of gluten. Specifically, we lack insight into the chemical structures of peptides formed from in vivo digestion that may stimulate the immune system. As a step toward filling this knowledge gap, we sought to analyze human urine by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Currently, LC–MS/MS is the most widely used technique for peptide sequencing in complex biological samples32. However, established LC–MS/MS methods suffer from technical limitations when applied to urinary peptidome analysis. High concentrations of urinary salts and metabolites, which are not efficiently removed by standard reversed-phase or liquid-liquid extraction procedures, can overwhelm chromatography systems and interfere with peptide detection33,34. Here, we develop a sample preparation and LC–MS/MS method that utilizes mixed cation exchange solidphase extraction to exclude these interfering molecules in a single step. This workflow overcomes problems we initially encountered with adapting established methods for urinary peptidomics, such as the need for time-consuming strong cation exchange purification and/or limited depth of peptide sampling. With it, we can now efficiently identify dietary gluten peptides and report the precise sequences of such peptides in the urine of human volunteers. We also undertake an exploratory clinical study, which revealed wheat-derived peptides that are substantially different in their chemical and biological properties and are differentially found in patients with CeD versus healthy controls. These peptides are attractive candidates for improving CeD diagnosis and for monitoring patient compliance to GFDs. They also set the stage for elucidating mechanisms underlying the anomalous ADME characteristics of gluten and other dietary proteins. More generally, the successful application of our urinary peptidomic workflow to CeD suggests it should be broadly applicable for the direct measurement of both endogenous and exogenous peptides present in urine. Results LC–MS/MS method development. Previous chromatographic and antibody-based detection methods suggested the presence of gluten-derived peptides in human urine but were incapable of directly elucidating their sequences25–29. To determine if an unbiased LC–MS/MS approach could be used for this purpose, we initially analyzed urine samples from volunteers who had consumed a meal rich in dietary gluten by following an extraction and analysis protocol typically used in LC–MS/MS-based proteomics studies (Supplementary Methods). These exploratory efforts yielded just one gluten-derived peptide identification. Furthermore, very few endogenous human peptides were detected (Supplementary Fig. 1). In accordance with challenges previously reported with urinary peptidome experiments33,34, we noted severe degradation of the LC column and MS electrospray source over consecutive analyses, preventing us from analyzing multiple samples without unacceptable interruptions in instrument operation. We, therefore, endeavored to systematically optimize each step in our peptidome extraction and LC–MS/MS analytical protocols in order to reliably detect peptides from urine. Starting with urine from an individual challenged with dietary gluten, we first confirmed it had measurable gluten levels by ELISA. The commercially available R5 monoclonal antibody we used targets the pentameric motif QXP(W/F)P found in many gluten proteins35 and is therefore expected to react with gluten peptides produced by patients with CeD and healthy controls. Using this reference specimen, we tested a variety of methods to enrich and purify urinary peptides. In addition, we optimized the LC gradient and MS acquisition method (Supplementary Methods). These efforts resulted in 29and 13-fold increases in the number of identifiable wheatand human-derived peptides, respectively, while eliminating longstanding problems with instrument contamination by background metabolites (Supplementary Methods, Supplementary Figs. 2 and 3, see also Method Specificity section below). Importantly, by combining solid-phaseextractiononamixedcationexchangecolumn with online reversed-phase LC and high-resolution MS/MS ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 2NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications analysis, this workflow shortened our sample preparation time from 2–3daysto6h(Fig.1). This allowed sufficient throughput for us to undertake a comparative analysis of clinical urine samples. Pilot identification of chemically defined urinary wheat peptides. To evaluate our optimized urine peptidome extraction and LC–MS/MS method, four healthy participants were recruited into a pilot study outlined as follows (Fig. 2a): On the first day, participants initiated a GFD. The GFD was maintained on the second day, and a pooled urine sample was collected over 8 h. On the third day, participants underwent a gluten challenge comprised of two wheat bagels (~18 g gluten), and subsequently collected a second pooled urine specimen over the next 8 h. LC–MS/MS analysis of these urine samples yielded an average of 24 unique peptide sequences per participant mapping to the wheat proteome, all of which were identified only in the postgluten challenge samples (Supplementary Dataset 3). The number of unique wheat peptides varied widely between individual volunteers (Fig. 2b). In contrast, the number of detected human peptides was essentially unchanged in the GFD versus postchallenge samples (Supplementary Fig. 4). In anticipation of recruiting larger cohorts of human participants, we sought to confirm our initial findings while also simplifying the dietary gluten challenge protocol. The four original participants, along with four additional healthy participants, were asked to fast overnight in place of the 48 h GFD employed in the pilot study (Fig. 2c). Then, a single urine sample was collected before consumption of two wheat bagels for breakfast and pooled urine collection over the next 8 h. Wheat peptides were readily detected in all post-challenge samples (Fig. 2d and Supplementary Dataset 4), while the number of human peptides was similar in both sample groups (Supplementary Fig. 5). These results support our initial finding that a dietary gluten challenge consistently leads to urinary gluten peptides that are measurable by LC–MS/MS. They also indicate that levels of these peptides fall to undetectable levels after overnight fasting, justifying the use of the abbreviated protocol for subsequent studies. Considering all sequences of the wheat-derived peptides identified from healthy participants (Supplementary Datasets 3 and 4), four noteworthy characteristics were apparent (Table 1). First, although 123 unique peptide sequences were identified, only one peptide was found in common in all 8 participants: GQQQPFPPQQPYPQPQPFPS. Its C-terminally truncated derivative, GQQQPFPPQQPYPQPQPFP, was identified in 7/8 participants; other overlapping variants of these peptides were also detected in more than one urine sample. While these sequences from α-gliadin proteins are not known to be recognized by patient-derived T cells, multiple studies have demonstrated that they can stimulate an innate immune response15–21. Strikingly, these prevalent urinary peptides do not have a motif that is recognized by monoclonal antibodies commonly used for gluten detection35,36. Thus, they would evade detection by traditional immunoassays. Second, we identified many peptides harboring known CeDrelevant T-cell epitopes37. The DQ2.5-glia-γ4c/DQ8-glia-γ1a epitope was most common, with 32 distinct peptides harboring the corresponding QQPQQPFPQ sequence. The DQ2.5-glia-γ5epitope (QQPFPQQPQ) also appeared frequently, as it was identified in 14 unique peptides. Other known T-cell epitopes, including DQ2.5glia-γ1/DQ8.5-glia-γ1/DQ8-glia-γ2 (PQQSFPQQQ), DQ2.5-gliaγ3/DQ8-glia-γ1b (QQPQQPYPQ), and DQ2.5-glut-L1/DQ2.2glut-L1 (PFSQQQQPV) were identified less frequently (Supplementary Table 1). Some T-cell stimulatory peptides, such as FLQPQQPFPQQPQQPYPQQPQQPFPQ and SQQPQQPFPQPQ QQFPQPQQPQ, harbor more than one epitope and were previously identified as being both proteolytically resistant and normalize loading 30 µmol creatinine ≈1-10 mL urine proteins, peptides, metabolites, salts block Cys reduction (DTT) alkylation (IAM) deplete proteins 10 kDa MWCO 2% formic acid discard proteins mixed cation exchange solid phase extraction discard metabolites/salts dry, reconstitute peptides (25 µL) LC-MS/MS on Orbitrap Fusion Lumos 70 min gradient (C18-RP) DDA OT MS/MS thousands of peptides identified in a single run urine sample 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Time (min) 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 64.28 27.09 64.33 62.29 60.97 60.91 27.18 60.88 56.80 64.43 60.37 69.60 27.82 69.64 32.87 50.96 64.50 64.62 51.01 46.63 33.06 68.20 51.04 69.73 39.09 27.92 43.62 33.69 20.72 24.45 28.57 55.30 71.40 75.05 12.23 20.12 10.41 75.29 83.61 6.23 6.83 2.45 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 913.4781 z=1 743.3941 z=1 755.4108 z=2 1180.6373 z=1 1381.7540 z=1 1016.5050 z=1 686.3726 z=1 1267.6703 z=1 808.4351 z=1 1096.4949 z=2 971.5003 z=1 1459.7382 z=1 312.1554 z=? 512.2729 z=1 1344.7213 z=1 425.2390 z=? 869.4445 z=? 596.3391 z=? Fig. 1 An efficient chemical extraction and LC–MS/MS workflow for urinary peptidomic analysis. Urine sample volumes are first normalized based on creatinine measurement. To prevent the formation of intraor intermolecular disulfide bonds that would confound downstream data analysis, cysteine (Cys) residues are reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAM). Proteins are depleted using a centrifugal filtration device with a molecular weight cutoff (MWCO) of 10 kDa under acidic denaturing conditions. The filtrate is processed using a mixed cation exchange solid-phase extraction column to remove metabolites and salts that interfere with LC–MS/MS analysis. After extensive washing, peptides are eluted from the column with methanol containing 5% ammonium hydroxide. Eluted peptides are dried in a centrifugal vacuum concentrator and reconstituted in water. Peptides are separated by nano-liquid chromatography (LC) on a reversed-phase column (RP-C18) and analyzed by tandem mass spectrometry (MS/MS) on an Orbitrap Fusion Lumos mass spectrometer operated in the data-dependent acquisition (DDA) mode, with precursor and fragment ions analyzed in high resolution in the Orbitrap (OT). The resulting LC–MS/MS data are searched against the desired databases (e.g., the human and wheat proteomes). The experimental workflow takes ~6 h to complete and results in the identification of thousands of peptides. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications 3 highly inflammatory to CeD patients through in silico and in vitro analysis38. Third, some peptides such as PyrQTFPHQPQQQVPQPQQ PQQP had undergone post-translational modification via cyclization of an N-terminal Gln into a pyroglutamic acid residue. The possibility that this modification occurs during sample processing cannot be excluded. However, N-terminal pyroglutamination is known to protect peptides from proteolytic degradation39–41 and thus may contribute to their stability in circulation. Last, we found gluten peptides from human urine that have not been described by any prior study as either being resistant to gastrointestinal digestion or as having any pathophysiological characteristics. One such peptide, SCHVMQQQCC, is derived from γ-gliadin and from the low-molecular-weight subunits of glutenins B and C. It was identified in 6/8 participants, while its C-terminally extended form was detected in 5/8 participants. Longer and shorter versions were also detected in multiple samples. This result illustrates the power our untargeted approach has for identifying wheat-derived peptides that have eluded prior description despite their seemingly common representation. Method specificity. To our knowledge, untargeted detection of diet-derived peptides in humans had not been reported prior to our proof-of-concept studies above. We, therefore, sought to confirm that our LC–MS/MS method reliably detects these peptides and accurately identifies their sequences in the complex background of the human urinary peptidome. To do so, we took advantage of the fact that rye and barley also contain proteolytically resistant gluten proteins42. For example, wheat gliadins (one of two major protein families that comprise gluten) are homologous to secalin proteins in rye and hordeins in barley (Fig. 3a). We thus anticipated that urine analysis should distinguish the particular grain consumed by volunteers who ate meals rich in either wheat, barley, or rye. To test this hypothesis, two healthy participants fasted overnight, and then, on different days, maintained a grain-free diet, or ate breakfasts prepared with approximately 1.5 cups of wheat, barley, or rye flour. Urine was then collected for 8 h, analyzed by our optimized LC–MS/MS protocol, and the data were searched against a customized database that included the human, rye, wheat, and barley proteomes (see “Methods”section). When participants maintained a grain-free diet, only a single grain peptide (likely a false discovery) was identified. In contrast, subsequent to wheat, rye, and barley dietary challenges, we respectively detected 51, 43, and 37 unique grain peptide sequences (Supplementary Dataset 5). The human peptide repertoire was similar in all specimens (Supplementary Fig. 6). Notably, all identified peptides were restricted to just one dietary challenge (Fig. 3b), suggesting that our method readily differentiates peptides formed from different dietary proteins. Indeed, when the sequences were mapped onto the grain proteomes, the majority were predominately derived from the corresponding dietary challenge (Fig. 3c and Supplementary Fig. 7). Our observation that fewer sequences could be mapped to the rye proteome from rye challenge urine is a likely artifact of the size of the available rye proteome: the UniProt resource contains ~100× fewer sequences than the wheat or barley proteomes, despite their genomes having similar sizes. Our data suggest that many of the detected sequences in the rye challenge urine may be derived from yet unannotated rye proteins whose sequences are also present in the wheat proteome. Taken together, these results demonstrate that the peptides detected by our method originate from the diet. They also confirm the specificity of our method to distinguish closely related dietary components. Preliminary analysis of banked urine from patients with CeD. To test our ability to identify wheat-derived peptides in the urine of individuals with CeD, we analyzed a set of nine urine samples banked from a prior study27. These urine samples were collected from single voids of patients with confirmed CeD diagnoses and were previously assayed for gluten presence by lateral flow immunoassay. This immunoassay used G12 and A1 monoclonal a c Diet Fast Gluten Human Volunteers Time (h) 0 spot urine dietary gluten challenge pooled urine ~10 ~18 0 10 20 30 40 Gluten Free Diet Gluten Challenge # Wheat Peptide Sequences Diet Gluten-Free Gluten Human Volunteers Time (h) sample extraction LC-MS/MS analysis 024 4832 56 pooled urine dietary gluten challenge pooled urine b d 0 10 20 30 40 50 # Wheat Peptide Sequences Overnight Fast Gluten Challenge sample extraction LC-MS/MS analysis Fig. 2 LC–MS/MS enables the identification of chemically defined dietary wheat peptides in human urine. a Pilot study design. bNumber of wheatderived peptide sequences detected in the urine of 4 healthy participants (4 males, 24-28 years of age) after a 24-h gluten-free diet and after a dietary challenge with wheat gluten. Horizontal line represents median. cSimplified study design. dNumber of wheat-derived peptide sequences detected in 8 healthy participants (5 males, 3 females, 24-28 years of age) after an overnight fast and after a dietary challenge with wheat gluten. Horizontal line represents median. In all LC–MS/MS experiments, peptide identifications were controlled at a false discovery rate of 1% using the PEAKS software decoyfusion approach59. Source data are provided as a Source Data file. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 4NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications Table 1 Examples of wheat-derived peptide sequences identified in human urine. Wheat peptide sequenceaUniprot protein accession(s)bProtein name(s) Mass (Da) # of participants with peptide GQQQPFPPQQPYPQPQPFPS GDA0_WHEAT; GDA1_WHEAT; GDA2_WHEAT; GDA9_WHEAT α/β-gliadin; α-gliadin 2292.0962 8 GQQQPFPPQQPYPQPQPFP GDA0_WHEAT; GDA1_WHEAT; GDA2_WHEAT; GDA9_WHEAT α/β-gliadin; α-gliadin 2205.0642 7 SQQPEQTISQQPQQPFPQQPHQPQQPYPQQQPYGSSL A0A290XYU3_WHEAT; U5UA46_WHEAT; R9XT67_WHEAT; R9XUY1_WHEAT ω-gliadin 4284.0259 6 SCHVMQQQCC GDB1_WHEAT; GLTC_WHEAT; GDB3_WHEAT; GLTB_WHEAT γ-gliadin; LMW glutenin 1336.4781 6 LGQQQPFPPQQPYPQPQPFPSQQP GDA1_WHEAT; A0A0K2QJX_WHEAT; A0A0E3Z7F7_WHEAT, A0A1K0JNE4_WHEAT α/β-gliadin; α-gliadin 2758.3500 5 SQQPQQPFPQQPHQPQQPYPQ B6ETR9_WHEAT; A0A290XYU3_WHEAT; U5UA46_WHEAT; R9XT67_WHEAT ω-gliadin; LMW glutenin 2512.1882 5 SCHVMQQQCCQ GDB1_WHEAT; GLTC_WHEAT; GDB3_WHEAT; GLTB_WHEAT γ-gliadin; LMW glutenin 1464.5367 5 PyrQTFPHQPQQQVPQPQQPQQP GDB2_WHEAT; B6DQB5_WHEAT; R9XWD0_WHEAT; R9XUB9_WHEAT γ-gliadin 2348.1296 5 PQQPPFSQQQQQQQQQQQPPFSQQQQPVL Q5MFQ2_WHEAT; D0EVP4_WHEAT; Q5MFQ1_WHEAT; Q5MFQ6_WHEAT LMW glutenin 3457.6763 3 TQQPQQPFPQQPQQPFPQQPQQPFPQ R9XUS6_WHEAT; A0A290XZ20_WHEAT; A0A290XZ51_WHEAT; A0A290XZ34_WHEAT γ-gliadin 3098.4998 3 TQQPQQPFPQQPQQPFPQTQQPQQPFPQ GDB2_WHEAT; B6DQB5_WHEAT, R9XV87_WHEAT; R9XWD0_WHEAT γ-gliadin 3327.6060 3 QPFPPQQPYPQPQPFP GDA0_WHEAT; GDA1_WHEAT; GDA2_WHEAT; GDA9_WHEAT α/β-gliadin; α-gliadin 1891.9257 2 P[I/L]QPQQPFPQQPQQPFPQPQcB6ETR9_WHEAT; A0A290XYW6_WHEAT; R9XT67_WHEAT; R9XUY1_WHEAT ω-gliadin 2354.1807 2 FLQPQQPFPQQPQQPYPQQPQQPFPQ GDB0_WHEAT, R9XV78_WHEAT; Q9FS77_WHEAT, B6UKM8_WHEAT γ-gliadin 3145.5410 1 CHVMQQQCCQdGDB1_WHEAT; GLTC_WHEAT; GDB3_WHEAT; GLTB_WHEAT γ-gliadin; LMW glutenin 1393.5000 1 PyrQQQQPPFSQQPPISQQQQPPFSQQQQPQF GLTA_WHEAT, D2DII3_WHEAT, Q6SPZ1_WHEAT, Q6SPZ3_WHEAT LMW glutenin 3416.6174 1 aResidues in bold indicate CeD-relevant T-cell epitopes, as defined in ref. 37. All Cys residues were detected in their carbamidomethylated forms due to derivatization with iodoacetamide during sample workup. “PyrQ”indicates glutamine residues that had undergone cyclization into pyroglutamic acid. bFor peptides mapping to more than four proteins in the UniProt database, only the first four accession codes are displayed. All identified peptide sequences and full details relating to peptide identification in each participants’urine are provided in Supplementary Datasets 3 and 4. cThe UniProt database contained otherwise identical peptides containing Leu and Ile at the same position. These two amino acids are isobaric and indistinguishable by our MS/MS method and thus the sequence is reported as [I/L]. dThe Met in this peptide was detected in its oxidized form. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications 5 antibodies, which specifically recognize the amino acid motifs QPQLP(Y/F) and QLP(Y/F)PQP, respectively36. Samples (n=4) from patients reporting adherence to a GFD and in which the measured gluten peptide concentrations were below the lateral flow assay limit of quantification were analyzed as negative controls. In accordance with the lateral flow results, we did not measure peptides mapping to the wheat proteome by LC–MS/MS. In contrast, all samples (n=5) for which lateral flow assays indicated high apparent gluten peptide concentrations, yielded at least 17 wheat peptide sequences (Fig. 4)byLC–MS/MS. The mean number of identified wheat peptides (74 per sample) was much higher than those described in our proof-of-concept studies with healthy participants (24 per sample; Fig. 2b, d) while the number of human peptides was similar (Supplementary Fig. 8). Strikingly, one sample from a patient with CeD yielded 206 distinct wheat peptides, a quantity four times greater than in any healthy participant analyzed previously. These intriguing observations led us to hypothesize that the urinary wheat peptide repertoires of patients with CeD are much more diverse than nonCeD individuals. We, therefore, initiated a prospective clinical study to formally test this hypothesis, described below. Clinical study. Adult patients with gastrointestinal symptoms including dyspepsia, bloating and diarrhea who were eating gluten-containing diets and were undergoing evaluation for CeD were recruited within the Celiac Disease Program at the Stanford Digestive Health Center, along with gender-matched healthy controls (Fig. 5a). CeD vs. non-CeD diagnoses were not known at the time of enrollment and were subsequently established by serology and endoscopy with biopsy. All participants undertook a dietary gluten challenge and urine collection according to the optimized study design depicted in Fig. 2c. At the end of the recruitment period, urine samples were processed and analyzed by LC–MS/MS. Compared to healthy controls (n=8) and patients ultimately diagnosed with non-celiac gastrointestinal disorders (n=5), patients with CeD (n=6) had approximately 5 times more unique gluten peptides in their urine (Fig. 5b), while the number of endogenous human peptides was similar in all three groups (Supplementary Fig. 9). Consistent with our pilot studies, variants of the innate immune response stimulating-peptide GQQQPFPPQQPYPQPQPFPS were detected in all individuals, regardless of clinical status (Supplementary Dataset 7). We analyzed whether patients with CeD had increased diversity of peptides with T-cell epitopes known to stimulate the adaptive immune response. Indeed, the number of peptides with at least one CeD-relevant T-cell epitope was Fig. 3 LC–MS/MS analysis of urine from two healthy participants challenged with dietary wheat, rye, and barley. a Illustration of sequence differences between closely related gliadin, secalin, and hordein proteins from wheat, rye, and barley, respectively. The boxes highlight sourcespecific sequence regions. bAll possible intersections of the three sets of peptide sequences identified after the grain challenges are depicted as an UpSet plot60. The individual set size is plotted to the left of each row, with the black portion of the bar representing sequences that map uniquely to the respective dietary challenge proteome, and the orange portion representing those that do not. There are no intersections between these sets of peptides, suggesting the peptides depicted in orange are nonetheless likely to have originated from the specific grain used for the dietary challenge. cNumber of distinct peptide sequences uniquely mapping to the wheat, barley, and rye proteomes following dietary challenges. Sequences that mapped to more than one of the proteomes were excluded from this figure. Sequences that mapped to more than one of the proteomes were excluded from this figure. In the rye diet, sequences mapping uniquely to the wheat proteome were only found in the rye diet, suggesting they are yet unannotated rye peptides. A full list of observed peptide sequences and their respective proteome mappings is provided in Supplementary Dataset 5. Participants in this experiment were males, ages 27 and 28. Source data are provided as a Source Data file. 0 50 100 150 200 # Wheat Peptide Sequences by LC-MS/MS Apparent Gluten Peptide Concentration by Lateral Flow Assay <6.3 ng/µL >100 ng/µL Fig. 4 LC–MS/MS analysis of banked urine samples from patients with CeD27.Urine collected from a single void was tested for the presence of gluten by an A1 and G12 monoclonal antibody-based lateral flow assay. Each data point represents a single urine sample, and the horizontal bar indicates the median. Information associated with the identification of these peptide sequences by LC–MS/MS is provided in Supplementary Dataset 6. Source data are provided as a Source Data file. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 6NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications approximately 4-fold higher in patients with CeD (Fig. 5c). CeDrelevant T-cell epitopes occurred more frequently in participants with CeD, and seven different T-cell epitopes completely absent in controls were found in patients (Table 2and Supplementary Table 2). Overall, we measured 289 unique wheat peptides from patients with CeD that were not detected in healthy individuals or patients with non-celiac gastrointestinal disorders, whereas only 37 peptides were shared between all three groups (Fig. 5d and Supplementary Dataset 7). To gain insight into the diversity of peptide sequences found in patients with CeD, we ranked the frequency by which CeDspecific peptide sequences were detected (Supplementary Dataset 8). Just four peptides were found in the majority of (≥4/6) patients (Table 3), whereas most (201) were detected only in single individuals. To understand the chemical space occupied by the peptides that were found in patients with CeD, we aligned the detected peptide sequences to the wheat proteome (Fig. 5e). Strikingly, patients with CeD not only had more variants (i.e., slightly longer or shorter versions) of peptides found in controls, but they also possessed peptides that mapped to distinct regions of the wheat proteome (Fig. 5f–h and Supplementary Fig. 10). For example, three patients had peptides spanning residues 194–199 of γ-gliadin (GDBX) that were absent in controls. Taken together, these results demonstrate that patients with CeD have peptidomes that are more diverse in their chemical and biological properties compared to control participants. Discussion Despite extensive research into the mechanisms by which gluten proteins play a role in CeD pathogenesis (Fig. 6), the identities of the actual diet-derived molecules that interact with the human 1262 GQQQP F P P Q KM Residue Number alpha-gliadin reference sequence detected unique peptide sequences sequence alginment and mapping Max Frequency 3 normalized to max frequency across all subjects Healthy Controls (No GI Complaints) (n=8) Patients Under Evaluation for CeD (n=11) serology endoscopy CeD Patients (n=6) Other GI Patients (n=5) 0 50 100 150 200 CeD Patients Healthy Controls # Wheat Peptide Sequences Other GI Patients * *ns 0 20 40 60 CeD Patients Healthy Controls Other GI Patients * *ns # Wheat Peptide Sequences with T-cell Epitopes ab c e d f gh CeD Patients 289 Other GI Patients 5 Healthy Controls 16 20 68 1 37 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications 7 immune system in the gut and other organs have been a longstanding mystery. As a step toward illuminating these molecular interactions, we developed a LC–MS/MS-based assay that directly detects the metabolic products of food grains. In contrast to in vitro gluten preparations that have been standard research tools in the CeD field for several decades, our method is compatible with in vivo dietary challenge. It therefore can reveal the precise chemical structures of dietary peptides that may drive CeD in humans. Furthermore, unlike traditional antibody reagents, the untargeted LC–MS/MS readout readily distinguishes the precise amino acid sequences of digestion-resistant peptides from wheat, barley, and rye. Thus, this method provides a unique window into gluten’s ADME characteristics and immunogenicity. Indeed, we observed more varied wheat peptide repertoires from patients with CeD than was previously appreciated –and ones that were much more diverse than those found in non-CeD individuals. Initially, our efforts were hindered by LC–MS/MS methods that were unsuited to the unique challenges of urinary peptidome analysis. Urine contains high concentrations of metabolites and salts that are difficult to separate from peptides and are incompatible with LC–MS/MS33,34. Therefore, we developed an extraction technique to remove these interfering compounds. This allowed us to achieve our main goal of wheat peptide identification, while also improving our ability to measure urinary peptides originating from other endogenous and dietary sources (Supplementary Fig. 3). Our workflow (Fig. 1) is compatible with standard reversed-phase LC–MS/MS instrumentation available in most proteomics laboratories32. Compared to published approaches employing solid-phase extraction techniques, our method identifies approximately 2 to 10 times more endogenous human peptides from typical 1–10 mL urine samples33,34,43,44. Moreover, our sample preparation technique requires only ~6 h, facilitating sufficient throughput for us to undertake a comparative analysis of the wheat-derived urinary peptidomes of patients with CeD and healthy controls. Given its high specificity (Fig. 3), our workflow has potential utility for other studies requiring analysis of the urinary peptidome. Although here we focused on the identification of wheat-derived peptides, we also identified over 30,000 human peptides (Supplementary Datasets 1–7), which is to our knowledge, is the largest collection of urinary peptides sequenced by LC–MS/MS to date. Parenthetically, we note that previous surveys of the urine peptidome found that hydroxyproline-modified collagen peptides were among the most abundant33,34,43,44. However, here we did not allow hydroxyproline as a variable modification in our database searches, as this modification was not relevant to our overriding goal of detecting biologically relevant, wheat-derived peptides. Therefore, we have deposited our raw data in the PRIDE database to facilitate the identification of additional peptides (e.g., by including other variable modifications or using alternative search engines). This study has identified, for the first time, the specific amino acid sequences and post-translational modifications of peptides resulting from in vivo digestion of gluten. In our pilot identification of dietary wheat peptides in healthy participants (Fig. 2), we identified sequences with known CeD relevance, as well as others that had never been implicated in CeD or identified as being resistant to gastrointestinal digestion. These findings underscored the value of our untargeted LC–MS/MS method and motivated us to analyze the urine of patients with CeD. Using banked urine specimens, we found that wheatconsuming patients with CeD had substantially greater gluten peptide diversity than non-CeD individuals (Figs. 2and 4). We confirmed this finding with a subsequent controlled clinical study (Fig. 5) in which participants followed our well-defined gluten challenge protocol (Fig. 2d). Mechanistically, this peptide Fig. 5 The urinary wheat peptidomes of patients with CeD are significantly more diverse than in healthy controls or patients with non-celiac gastrointestinal disorders. a Clinical study design. Participants were recruited over approximately 2 years within the Celiac Disease Program at the Stanford Digestive Health Center. Participant characteristics are reported in Supplementary Tables 3 and 4. bNumber of wheat peptides detected in pooled urine samples collected for 8 h subsequent to a dietary challenge with two bagels (~18 g gluten). Patients with CeD had substantially more wheat peptide sequences compared to healthy controls (p=0.017) or patients with non-celiac gastrointestinal disorders (p=0.017). cNumber of unique wheat-derived peptide sequences with at least one T-cell epitope in patients with CeD compared to healthy controls (p=0.019) and patients with non-celiac gastrointestinal disorders (p=0.046). b,cStatistics were derived from n=6 patients with celiac disease, n=5 patients with non-celiac gastrointestinal disorders, and n=8 healthy controls using a one-way Kruskal–Wallis ANOVA/Dunn’s multiple comparison test; horizontal bar represents the median. dVenn diagram comparing the unique peptide sequences detected in healthy controls, patients with CeD, and other GI patients. A full listing of peptide sequences is provided in Supplementary Dataset 7. eSchematic describing how the frequencies at which urinary wheat-derived peptides map to proteins in the wheat proteome were plotted as heatmaps. f–hHeatmap representation of detected peptide sequences in alpha/beta gliadin 2 (GDA2), γ-gliadin 2 (GDB2), and gamma-gliadin X (GDBX) demonstrates that while many peptides map to the same region of the wheat proteome, the urinary wheat peptidomes of patients with CeD also occupy distinct chemical space. Below the maps, the locations of CeD T-cell epitopes are highlighted in red, and the relative density of Gln and Pro is indicated in yellow. Heatmaps for other proteins in the wheat proteome are provided in Supplementary Fig. 10. In b–h, all samples were analyzed by LC–MS/MS in duplicate and the aggregated results are shown. Analyses of individual replicates are provided in Supplementary Fig. 11. Full details on LC–MS/MS identification of peptide sequences are provided in Supplementary Dataset 7. Source data are provided as a Source Data file. Table 2 Number of detected T-cell epitopes in patients with celiac disease (CeD), non-celiac gastrointestinal disorders (GI), and healthy controls (HC). Epitope name(s)aEpitope sequence CeD GI HC DQ2.5-glia-α1a PFPQPQLPY 2 0 0 DQ2.5-glia-α1b PYPQPQLPY 1 0 0 DQ2.5-glia-α2 PQPQLPYPQ 2 0 0 DQ2.5-glia-α3 FRPQQPYPQ 1 0 0 DQ2.5-glia-γ1/DQ8.5-glia-γ1/ DQ8-glia-γ2 PQQSFPQQQ 1 0 1 DQ2.5-glia-γ3/DQ8-glia-γ1b QQPQQPYPQ 6 1 0 DQ2.5-glia-γ4a SQPQQQFPQ 1 0 0 DQ2.5-glia-γ4b PQPQQQFPQ 8 4 3 DQ2.5-glia-γ4c/DQ8-glia-γ1a QQPQQPFPQ 105 29 36 DQ2.5-glia-γ4d PQPQQPFCQ 2 0 1 DQ2.5-glia-γ4e LQPQQPFPQ 28 5 4 DQ2.5-glia-γ5 QQPFPQQPQ 37 10 15 DQ2.5-glia-ω1/DQ2.5-hor-1/ DQ2.5-sec-1 PFPQPQQPF 3 0 1 DQ2.5-glia-ω2 PQPQQPFPW 1 0 0 DQ2.5-glut-L1/DQ2.2-glut-L1 PFSQQQQPV 17 2 11 DQ2.5-glut-L2 FSQQQQSPF 1 0 0 DQ8.5-glut-H1/DQ8-glut-H1 QGYYPTSPQ 2 0 1 aEpitope sequences and nomenclature are defined in ref. 37. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 8NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications diversity may stem from the fact that individuals with active CeD have elevated intestinal permeability, which likely allows wheatderived peptides to more readily cross the epithelial barrier5. This phenomenon could lead to increased peptide concentrations in the systemic circulation and in urine, thereby enhancing their ability to be detected. A different (or complementary) explanation is that patients with CeD digest and/or metabolize wheat differently than other individuals. Such potential differences in wheat metabolism could contribute to the onset of CeD. Future analyses of patients with other non-CeD enteropathies known to cause “leaky gut”such as tropical sprue as well as disorders in which the mucosa remains grossly intact such as irritable bowel syndrome with diarrhea should be insightful in pinpointing the origins of the increased peptide diversity observed in patients with CeD45,46. More fundamentally, analysis of urine from individuals consuming diets with various food additives, such as microbial transglutaminase, should help to address the emerging hypothesis that these additives contain increased levels of immunogenic peptides and/or directly contribute to increases in intestinal permeability47,48. We identified 17 distinct immunotoxic epitopes known to be presented on MHC class II molecules and recognized by T cells that reside in the lamina propria of patients with CeD. Many of these epitopes occurred more frequently in patients with CeD than in controls (Table 2). Notably, however, we were unable to detect in its intact form an extensively studied and highly inflammatory 33-residue peptide from α2-gliadin that accumulates in the mammalian gut lumen under physiological conditions by virtue of its resistance to gastrointestinal proteolysis4. Its apparent absence from human urine could be due to physicochemical properties that hinder absorption, promote systemic metabolism, or make it unamenable to chemical extraction and/ or detection by mass spectrometry. Nonetheless, it is just as noteworthy that an analogous 26-residue peptide from γ-gliadin, FLQPQQPFPQQPQQPYPQQPQQPFPQ, was observed in 2/7 patients with CeD. This peptide is also proteolytically resistant and highly inflammatory to CeD-specific T cells on account of its polyvalency38. Despite the abundance of immunogenic wheat peptides in urine, thus far we have been unable to definitively identify any peptide that underwent regioselective Gln deamidation by transglutaminase 2 (TG2), an important step in CeD pathogenesis (Fig. 6). Because TG2-catalyzed deamidation increases the immunogenicity of gluten-derived peptides by several orders of magnitude4, it is possible that a relatively small molar fraction of absorbed gluten is deamidated in patients with CeD, making the concentrations of these peptide species too low to be detectable. Alternatively (or additionally), deamidation may enhance the metabolic lability of circulating gluten peptides. The possibility that their high affinity for HLA-DQ2 or -DQ8 alters their ADME characteristics should not be overlooked. Regardless, future development of targeted LC–MS/MS methods could facilitate the detection of low-abundance deamidated gluten peptides. There is increasing interest in non-invasive monitoring of GFD compliance via urinalysis26–28,49, as a large proportion of patients with CeD experience symptoms even when attempting to adhere to a GFD50. Available methods predominantly exploit the G12 monoclonal antibody, which preferentially recognizes the immunotoxic QPQLP(Y/F) motif found in several CeD-relevant T-cell epitopes36. Our data suggest that this motif is rare in urine from individuals who consume dietary wheat (Table 2). In contrast, the sequence GQQQPFPPQQPYPQPQPFPS appeared in wheat product partially digested wheat product wheat peptides proteases translocation translocation deamidation TG2 GI Tract Lamina Propria Blood Stream filtration (to be excreted) Kidneys Bowman’s Space Glomerular Capillaries Glomerulus Fig. 6 Schematic representation of relevant metabolic products of dietary gluten in humans. Wheat-derived proteins such as gluten are resistant to gastrointestinal proteolysis. Incomplete digestion preserves disease-causing T-cell epitopes in peptides that accumulate in the gut lumen. A portion of these gluten peptides translocate to the lamina propria where transglutaminase 2 (TG2) deamidates selected Gln residues (brown dots) in some sequences, thereby converting them into Glu residues (yellow dots). Absorbed peptides can also enter the bloodstream, and are further metabolized and/ or excreted in urine. Table 3 Wheat-derived peptide sequences detected in the majority of patients with CeD but not in control groups. Wheat peptide sequence Protein name # of CeD patients with peptide PQQQIPQQHQIPQQPQQFPQQ ω-gliadin 5 GQQQQQFPGQQQPFPPQQPYPQPQP α/β-gliadin 4 PELQQPIPQQPQQPFPLQPQQPFPQQ ω-gliadin 4 PTSPQQSGQGQQPGQWQQPGQGQPG HMW-glutenin 4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28353-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:888 | https://doi.org/10.1038/s41467-022-28353-1 | www.nature.com/naturecommunications 9