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Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine Susy Piovesana, Anna Laura Capriotti, Andrea Cerrato, Carlo Crescenzi, Giorgia La Barbera, Aldo Laganà, Carmela Maria Montone, and Chiara Cavaliere Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03034 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Analytical Chemistry

Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine

Susy Piovesana1, Anna Laura Capriotti1*, Andrea Cerrato1, Carlo Crescenzi2, Giorgia La Barbera3, Aldo Laganà1,4, Carmela Maria Montone1, Chiara Cavaliere1

1

Department of Chemistry, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185

Rome, Italy 2

Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, I-84084 Fisciano,

SA, Italy 3

Department of Nutrition, Exercise and Sports, University of Copenhagen, Nørre Allé 51, DK-2200

Copenhagen, Denmark 4

CNR NANOTEC, Campus Ecotekne, University of Salento, Via Monteroni, 73100 Lecce, Italy

*Corresponding author Department of Chemistry Università di Roma “La Sapienza” Piazzale Aldo Moro 5 00185 Rome, Italy

E-mail: [email protected] tel: +39 06 4991 3945

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Keywords Peptidomics; dipeptides; urine; HILIC; reversed phase; graphitized carbon black

Abstract

Short peptide sequences represent emerging analytes in a variety of fields, including biomarker discovery, but also a well-known analytical challenge in complex matrices, due to the low abundance, extensive suppression during MS analysis and lack of workflows, as they cannot be identified by ordinary peptidomics strategies and coverage is extremely limited by metabolomics as well. In this context, in this work, a solid phase extraction method was developed for the clean-up and enrichment of dipeptides, tripeptides and tetrapeptides in urine using graphitized carbon black Carbograph 4 as the sorbent. The method was first developed on analytical standards spiked in urine, with recoveries in the range 60-100%. Then the method was applied to urine samples from healthy volunteers. The enriched urine samples were analysed by ultra-high performance liquid chromatography (UHPLC) using an orthogonal strategy in which both a reversed phase (RP) C18 column and a zwitterionic hydrophilic interaction liquid chromatography (HILIC) column were used, for better coverage of peptide polarity and improved detection of peptides. High resolution mass spectra were acquired in data dependent mode using a suspect screening strategy with inclusion list; peptides were identified by a semi-automated workflow for feature extraction, candidate mass filtering and MS/MS spectra comparison with in-silico mass spectra. The complementarity of the orthogonal separation strategy was confirmed by peptide identification, resulting in 101 peptides identified from the RP runs and 111 peptides from the HILIC runs, with 60 common identifications. The method is applicable to both hydrophobic and hydrophilic peptides.


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Peptides were stable over 2 h after collection and protease inhibitors were not necessary, as no formation of artefacts was observed.



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Introduction

Short peptide sequences are emerging as promising analytes in different research fields. In this context, one of the most developed research fields is the discovery of new bioactive peptides in food or food by-products with health-promoting bioactivities, such as antioxidant, antihypertensive and antimicrobial properties1–4; short peptides can be investigated in food also as biomarkers for food traceability5. Another emerging and promising field is the identification of short peptide biomarkers of disease6, because peptides can have specific functions, for instance, in modulating cancer cells aggressiveness7. In the latter context, while peptide sequences longer than 4 amino acids can be investigated by application of proteomics technologies, small peptides (i.e., dipeptides, tripeptides and tetrapeptides) cannot be dealt with in such a way and are currently mainly investigated within untargeted metabolomics studies8,9. Although short peptides have long been considered the by-products of protein degradation by peptidases, still they may bear very important biological significance under pathological conditions. For instance, they could reflect the expression of aberrant proteolytic activity in different pathological states, including malignant tumors, such as in epithelial ovarian cancer10 and hepatocellular carcinoma11. Recently, dipeptides and tripeptides were specifically investigated in plasma as biomarkers of epithelial ovarian cancer and benign ovarian tumours10, suggesting to be promising diagnostic and prognostic biomarkers also in other diseases. Despite the potential of short peptides as biomarkers, their diagnostic and prognostic value is not well characterized, yet, as they are generally underinvestigated due to lack of dedicated analytical workflows. Direct analysis of peptides in complex biological matrices is not usually feasible due to the potential association with high abundant components, such as proteins, and due to the low abundance of peptides compared to other molecules, which can cause extensive ion suppression during electrospray ionization (ESI)12. Some of these issues can be overcome by a dedicated protocol for enrichment and clean-up of short peptides. However, sample clean-up is not


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straightforward either due to the heterogeneous nature of short peptides in terms of polarity, which hinders their pre-concentration from complex matrices and their chromatographic separation. In the latter regard, the commonly used reversed phase (RP) chromatography on C18 can efficiently separate only short peptides made up of hydrophobic residues. Alternative separation strategies have been considered to improve the coverage of polar short peptides, such as hydrophilic interaction chromatography (HILIC)13,14. Also, separation on polar C1815 or porous graphitic carbon16 has been investigated to find a compromise suitable to cover a wider range of analyte polarity. Other issues in short peptide analysis include the lack of automated systems for sequence identification from high-resolution mass spectra and tandem mass spectra (MS/MS). Indeed, ordinary bioinformatic software developed for proteomics cannot identify sequences shorter than 5 amino acids17, and metabolomics databases are currently not sufficiently complete to cover the 168,400 possible combinations of proteinogenic amino acids in dipeptides, tripeptides and tetrapeptides, let alone possible modifications or isomeric structures18. Only few papers specifically aim at identifying dipeptides, tripeptides and tetrapeptides without pre-column derivatization, with a number of applications in the field of bioactive peptide discovery in protein hydrolysates from food and wastes13,19,20 and limited investigations in biofluids10,21. Derivatization is a strategy used to improve the detection of amino acids and short peptides, and it is usually performed during sample preparation with tagging reagents (phenyl isothiocyanate, naphthalene-2,3-dialdehyde or dabsyl chloride), which were used for investigation of bioactive peptides in food22 and plasma23. In these protocols, a step dedicated to peptide derivatization is specifically performed prior to chromatography for targeted analysis of short peptides20 by quadrupole MS24, UV-vis detector22, fluorescence detector25 or peptide sequencer based on Edman degradation23. Due to its simple and non-invasive collection, urine is one of the most exploited biofluids for biomarker discovery by liquid-biopsy strategies in several diseases26–28, especially those directly



connected with the urinary tract, such as bladder cancer29,30. In this context, metabolomics studies indicated some short peptides as possible biomarkers; for instance, the tetrapeptide Asp-Asp-GlyTrp (DDGW) was upregulated in early bladder cancer urine samples, whereas the tetrapeptide GlyGly-Ala-Lys (GGAK) was shown to be downregulated30. While methods for peptidomic analysis of urine have been developed for peptides longer than 5 amino acids31,32, no strategy specifically meant for 2-4 amino acid long native peptides in urine has ever been described. In the present paper, a strategy for the identification of underivatized short peptides in urine is provided. The chromatographic separation of both hydrophilic and hydrophobic short peptides was achieved by ultra-high performance liquid chromatography (UHPLC) both by RP and HILIC; high resolution MS (HRMS) allowed the detection of the eluting peptides by data dependent mode using a suspect screening strategy with inclusion list, which allowed to fragment the precursor peptides and to identify them by a semi-automated workflow for feature extraction, candidate mass filtering and MS/MS spectra matching to in-silico mass spectra. Due to the complexity of the urine matrix and in order to increase the coverage of the low molecular weight window of the peptidome, an enrichment strategy was developed for isolation and clean-up of short proteinogenic peptides by graphitized carbon black (GCB) solid phase extraction (SPE). More specifically, Carbograph 4 was used, as it was previously demonstrated to be able to efficiently concentrate very polar compounds33,34 better than other commercial sorbent materials35. The method was developed on spiked urine samples and then applied to identification of short peptides in urine from healthy volunteers. Additional issues were also considered, namely peptide time stability after collection and artefacts formation due to protease activity.

Experimental Section

Chemicals and Materials


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Optima® LC-MS grade water, acetonitrile (ACN) and methanol (MeOH) were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Trifluoroacetic acid (TFA) was supplied by Romil Ltd (Cambridge). Formic acid and ammonium formate were purchased from SigmaAldrich (Germany). The synthetic peptides Gly-Asp-Leu-Glu (GDLE), Leu-Pro-Leu (LPL), Ile-ProPro-Leu (IPPL), Lys-His (KH), Pro-Ile (PI), Arg-Phe (RF), Ser-His (SH), Val-Glu-Pro (VEP), ValArg-Gly-Pro (VRGP), Pro-Leu (PL), Ile-Pro-Ile (IPI), Leu-Pro (LP), Lys-His- Lys (KHK) were purchased from Thermo Fisher Scientific (Ulm, Germany). Peptide stock solutions were prepared at 1 mg mL-1 concentration in H2O/TFA, 99:1 (v/v); two working solutions, one for RP and HILIC, were prepared from the stock solutions at 1 ng μL⁻1 by dilution in a mixture matching the composition of the mobile phase at gradient start.

Preparation of Urine Samples and Peptide Extraction

The first urine of the day was collected from 10 healthy volunteers fasting after midnight. The collected urine was pooled, centrifuged at 1000 × g, acidified with HCl to pH 2, aliquoted and stored at -20 °C until further processing. Urine aliquots were thawed at room temperature and centrifuged at 8000 × g to remove any insoluble debris. The procedure for GCB SPE was first developed on pure standards and spiked urine samples, as summarized in Table S1. The recovery from standard solutions were calculated as ratio between the SPE eluate and pure standards in resuspension mixture at the expected nominal concentration. Recoveries from spiked urine samples, matrix effect and process efficiency were calculated as extensively described previously36 (experimental details are reported in the Supplementary material). For the combined SPE and clean-up of short peptides, in the final procedure a cartridge packed with 500 mg Carbograph 4 (Lara S.r.l., Formello, RM, Italy) was used. The cartridges were prepared by manually packing the GCB sorbent into 6 mL polypropylene tubes (Sigma-Aldrich): a polypropylene frit (Sigma-Aldrich) was placed at the bottom of the cartridge then, working on an ACS Paragon Plus Environment


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analytical balance (E50S by Gibertini Elettronica, Milan, Italy), 500 mg of Carbograph 4 bulk material (130 m2/g surface area, 20/400-120/200 mesh size) was added into the cartridge, which was finally blocked with an additional upper polypropylene frit to avoid dispersion of the sorbent during all operations. For SPE, the adsorbent was previously washed with 5 mL of CH2Cl2/MeOH, 80:20 (v/v) with 20 mmol L-1 TFA and 5 mL of MeOH with 20 mmol L-1 TFA. Then, the material was activated by flushing with 10 mL of 0.1 mol L-1 HCl and finally conditioned with 10 mL of 20 mmol L-1 TFA. Then the urine sample was loaded (2 mL urine diluted to 10 mL with 20 mmol L-1 TFA) and the cartridge washed with 2 mL of 20 mmol L-1 TFA and 0.5 mL MeOH (used to remove the cartridge dead volume). Finally, analytes were eluted by back flushing with 10 mL of CH2Cl2/MeOH, 80:20 (v/v) with 20 mmol L-1 TFA. The eluate was evaporated at room temperature in a Speed-Vac SC250 Express (Thermo Savant, Holbrook, NY, USA) and the residue reconstituted either in 200 µL H2O for RP separation or 200 µL of ACN/H2O, 75:25 (v/v) for HILIC separation. For dilute and shoot direct analysis, urine samples were 1:1 diluted with mobile phase and analysed as described in the following section. For the experiments evaluating the effect of protease inhibitors, 1 tablet cOmplete™, Mini, EDTAfree Protease Inhibitor Cocktail (Sigma-Aldrich) was added to 10 mL urine before centrifugation. For evaluation of collection time, the urine samples were left to stand at room temperature for 2 h before centrifugation and acidification. For all experiments, three experimental replicates were performed.

Ultra-High Performance Liquid Chromatography-HRMS Analysis

Recovery experiments on pure standards and spiked urine samples were performed by multiple reaction monitoring (MRM) on an Ultimate 3000 UHPLC system (Thermo Fisher Scientific) coupled with a triple quadrupole mass spectrometer, mod. TSQ Vantage EMRTM (enhanced mass range) via a heated ESI source. Chromatography was accomplished by RP separation as later


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described in this section, whereas the detailed parameters for MRM acquisition are reported in the supplementary material Table S2. The UHPLC Vanquish binary pump H (Thermo Fisher Scientific, Bremen, Germany), equipped with a thermostatted auto-sampler and a thermostatted column compartment, was used for analysis of native peptides in urine. Twenty μL of each sample were injected onto a Kinetex XB-C18 (100 × 2.1 mm, 2.6 μm particle size, Phenomenex, Torrance, USA) for RP separation or on a iHILIC®-Fusion UHPLC Column, SS (100 × 2.1 mm, 1.8 μm particle size, Hilicon, Umeå, Sweden) for HILIC separation. RP separation was performed as previously reported37; the C18 column was maintained at 40 °C and the elution was carried out with H2O (phase A) and ACN (phase B) both with 0.1% TFA (v/v) at 0.4 mL min-1. The chromatographic gradient was the following: 1% phase B for 2 min, 1–35% B in 20 min, 35–99% B in 3 min; at the end of the gradient, a washing step at 99% B for 3 min and a re-equilibration step at 1% B for 5 min were performed. HILIC separation was performed at 40 °C and 0.3 mL min-1. H2O with 0.1% formic acid and 10 mmol L-1 ammonium formate (phase A) and ACN (phase B) were used as mobile phase with the following gradient: starting from 95%, phase was B was lowered to 25% in 15 min, maintained for 10 min, and finally the column was equilibrated for 5 min at 95% B. The chromatographic system was coupled to a hybrid quadrupole-Orbitrap mass spectrometer Q Exactive (Thermo Fisher Scientific) using a heated ESI source. The ESI source was operated in positive mode under the following conditions: 220 °C capillary temperature, 50 (arbitrary units, a.u.) sheath gas, 25 (a.u.) auxiliary gas, 0 (a.u.) sweep gas, 3200 V spray voltage, 280 °C auxiliary gas heater temperature, 50 (%) S-Lens RF level. HRMS top five data dependent acquisition mode was performed in the range m/z 150–1000 with a resolution (full width at half maximum, FWHM, m/z 200) of 70,000. Automatic gain control (AGC) target value was 500,000 in full scan, with a max ion injection time set of 50 ms. Isolation window width was 2 m/z. Higher-energy collisional dissociation (HCD) fragmentation was performed at 40% normalized collision energy at resolution of 35,000 (FWHM, m/z 200). AGC target value was 100,000. Dynamic exclusion was set to 3 s. An



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inclusion list with the exact m/z values for singly charged precursor ions was used for data dependent acquisition. Raw data files were acquired by Xcalibur software (version 3.1, Thermo Fisher Scientific). Three technical replicates were performed for each experimental replicate. To ensure the stability and reproducibility of the HILIC column, the precaution of flushing the column with water at the end of sample analysis was taken to remove modifiers used for sample separation.

Data Analysis and Peptide Identification

Peptides were identified as previously described37. Xcalibur raw files were imported in the freeware MZmine 2 software (v2.37, http://mzmine.sourceforge.net/)38. Briefly, a list of ions for each scan was generated using a mass detection module. Then, a chromatogram was built from the mass list previously generated and it was deconvoluted into individual peaks. The isotopic peak grouper module was used to remove less abundant isotopic peaks. The peak lists obtained for samples and blanks (ultrapure water) were aligned to remove the peaks in common with the blank and keeping only repeatable features (peaks common to all three technical replicates). All possible amino acid combinations in di- tri- and tetrapeptides, without duplicate masses, were produced using Matlab R2018a (5095 combinations), and the exact m/z values for singly charged precursor ions were used to build a database. The features that matched with the home-made built database were further investigated by MS/MS spectra inspection. Only repeatable features (peaks common to all three technical replicates) were used for peptide identification by matching the experimental product ion spectra to the in-silico generated MS/MS spectra using mMass39. Peptide identifications were not validated by comparing the experimental retention time and MS product ions to those of a pure standard, therefore they are to be considered tentatively identified.

Results and Discussion


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Short Peptide Pre-Concentration Method

Short peptides made up of two, three or four amino acids can have a wide range of polarity, therefore their retention under typical RP conditions is poor for very polar sequences. For this reason, a clean-up strategy based on typical C18 is not feasible and different protocols must be pursued. In this regard, the GCB adsorbent was chosen as a suitable stationary phase for the cleanup of urine samples, due to its multiple interactions with a wide range of compounds. The GCB can work as a RP adsorbent and retain analytes by strong hydrophobic interaction, which is suitable for non-polar and polar compounds35. Moreover, the graphitic structure can contribute to retention both by electrostatic interaction, which occurs by polarization, and by π stacking interaction with aromatic moieties and H-π interactions40. Finally, GCB has the peculiar ability to retain anions as well, by electrostatic interactions with chromene-like heterogeneities on the GCB surface, which act as ion-exchange sites and are particularly suited for the retention of acidic and very acidic compounds41,42. As a whole, peptide clean-up on GCB would be suitable to maximize weak hydrophobic interactions with short peptides as well as interactions with charged side chains, charged C- and N-terminals, and aromatic residues, therefore making it a suitable strategy to enrich peptides and to remove salts in urine, as well as compounds with strong acid moieties, such as sulfate an glucoronate conjugated metabolites, which are highly abundant in urine and which would potentially be eluted only by an ion exchanger elution mixture41,42. At the beginning of method development, a peptide standard mixture was used to assess the recovery by different tested procedures (Table S1), following the general protocol depicted in Figure S1, which includes column preparation and conditioning, sample loading, one or two washing steps and one or two elution steps. More specifically, as far as the loading step was concerned, three loading buffers were tested, which varied for the acid modifier (formic acid, HCl or TFA). The acidic medium of the loading buffer was selected to cope with the poor solubility of some peptides in pure water. Additionally, given the reported presence of heterogeneities on the



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GCB structure with potentially reactive oxygen species, the loading buffer under acidic conditions would also avoid irreversible chemical bond by nucleophilic addition of amine moieties with groups potentially present on the surface, such as epoxides, as previously reported for graphene oxide43. Finally, and acidic medium was chosen to maximize retention from water. In fact, though the mechanistic details are outside the scope of the present paper, theoretical data on the interaction of amino acids with graphitic surfaces indicated a competing interaction of zwitterionic amino acids either with water or with graphene, with the former interaction preferred due to the better solvation of amino acid zwitterionic states. On the other hand, a strong interaction was reported between graphene and positively charged residues, a charge state which could be easily obtained for peptides by an acid modifier in the loading buffer40. Therefore, formic acid was originally tested and provided recoveries above 74% for most standard peptides except the very hydrophilic ones having Grand average of hydropathicity (GRAVY) values below -2 (KHK, KH, SH, Figure S2a, Experiment 1). The low recovery was not attributed to poor retention on GCB under the tested loading conditions, as checking the loading and washing fractions indicated only 20% loss for SH but no loss for KHK or KH. As far as the other standards were concerned, the retention was generally acceptable, with a 10% loss only for PL, PI, LP, RF and GDLE. Nevertheless, a change in loading conditions was tested to reduce analyte loss; HCl was tested as the acid modifier. HCl is probably the most employed modifier for analyte loading on GCB, and it is commonly used to activate the ion exchange sites on GCB materials. The use of a stronger acid than formic acid allowed to improve the recovery, which was above 76% for most peptides, included RF and GDLE which were partially poorly retained with formic acid, but not of PL, PI, LP, which were still not efficiently retained in the loading step (Figure S2a, Experiment 2). Therefore, a pairing acid was used to maximize hydrophobic retention on GCB. TFA was used for the purpose and indeed retention of peptides during loading was improved (Figure S2a, Experiment 3). This effect could be ascribed to intimate ion paring with TFA, which generates heavier, neutral and less hydrophilic species, thus enhancing their interactions with GCB. The phenomenon can be explained with a


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typical adsorption model by ion-pairing RP retention, as previously documented for the retention of peptides on C18 stationary phases44 and porous graphitic carbon45. Aside from the considerations on peptide retention on the GCB absorbent, the low recovery of the most hydrophilic and basic peptides was attributed to poor elution from GCB. This experimental observation is consistent with mechanistic data on the strong retention of positively charged short peptides on graphene40. In fact, positively charged residues, along with aromatic amino acids, represent the best residues for the interaction with graphene due to the right combination of big side chains, van der Waals contribution and π-π interaction of the aromatic moieties. As far as the positively charged species were concerned, a linear correlation was reported between the hydropathy index of the most interacting positively charged amino acids (Arg, Gln, Ans and Lys) and the interaction with the graphitic surface, which is attributed to the favorable coulombic interactions occurring with the negatively charged water oxygen found in the first water shell of waters interacting with the graphene surface. On the other hand, the negatively charged residues, such as Glu, display a reduced binding due to the repulsive interactions with these ordered waters. The model was also extended from amino acids to the tripeptide Arg-Gly-Asp (RGD)40. Given the above, the eluent composition was changed and tetramethylammonium chloride (TMAC) was added to the second elution step. TMAC is usually employed to elute compounds with strong acid moieties in the structure, such as sulfates, and allows elution by ion exchange46. An elution buffer consisting in CH2Cl2/MeOH, 80:20 (v/v) with 10 mmol L-1 TMAC (Table S1, Experiment 3) was tested for the purpose of eluting the most retained peptides. Nevertheless, the most polar species recoveries were not improved, thus implying extremely strong interactions between those species and GCB. To cope with this, in Experiment 4, peptides were back-flushing eluted using CH2Cl2/MeOH, 80:20 (v/v) with 10 mmol L-1 TMAC and 50 mmol L-1 formic acid, after removing water with 500 µL of MeOH to improve contact between the eluent and the GCB surface (Table S1, Experiment 4). Back-flushing elution has been widely used in the case of very intense interactions with carbonaceous materials, as it allows to reduce analyte path throughout the cartridge while



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conveniently reducing the volume of eluent. Indeed, back-flushing allowed peptides to be efficiently eluted from GCB, even in the case of the most retained ones (KH and KHK), which were never successfully eluted by the earlier procedures. In spite of a considerable improvement in terms of recoveries, the use of TMAC led to several issues due to its ion suppressing nature in ESI sources and a noticeable loss in reproducibility. The negative effect of residual TMAC after evaporation was evaluated by comparing peak areas for the spiked GCB eluent mixture with or without TMAC. Suppression was as extensive as a ten-fold decrease in MS signal intensity for some peptide standards. Since short peptides are very low abundant in body fluids samples, strong ion suppressing species such as TMAC were not considered suitable to our purpose. In Experiment 5 (Table S1), the same modifier employed in the loading buffer was tested in the elution buffer (20 mmol L-1 TFA). Aside from solving the reproducibility and ion suppression issues, TFA further enhanced peptide recoveries. Due to its ion paring and complexing nature, TFA could in fact facilitate peptide elution, by minimizing the electrostatic interactions and forming more hydrophobic complexes, which were more soluble in the elution buffer. Increasing the TFA concentration resulted in a significant loss in the recoveries, which could be due to ion suppression and acidic hydrolysis of the most polar peptides (Experiment 6, Table S1). After identification of the most suited conditions for standard peptide recovery, spiked urine samples were used to determine the efficiency of the developed clean-up and the possible matrix effect. The test was done using a 500 mg Carbograph 4 cartridge, to exclude possible saturation of the GCB sorbent due to other compounds in the urine sample (Table S1). Notably, the recoveries of standard peptides from spiked urine samples were nearly unaffected compared to the standard peptides from neat spiked solvents used in method development, and were in the range 60-100% (Table S3). The performance of the clean-up strategy using GCB was also evaluated by calculating the matrix effect, which resulted in an enhancement for 8 standards and a suppression for 5, in all cases within ±20%. The process efficiency was calculated from recovery and matrix effect data and was in the range 51% (for SH) and 113% (VRGP).


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Under the final conditions, the recovery of standard peptides both from water and spiked urine samples resulted independent from the hydrophilicity of the peptide (Figure S3a), which is a valuable behavior making the developed method suitable for isolation and pre-concentration of both hydrophilic and hydrophobic short peptides and which would be difficult to achieve with different sorbents. As far as the molecular weight was concerned, the study from standard recovery indicated a trend, with recoveries increasing with the molecular weight of the peptides (Figure S3b). Finally, a trend was observed for the pI as well. The largest recoveries were obtained for acidic and basic peptides (Figure S3c), whereas peptides with pI close to neutrality had the lowest recovery. In any case the recovery was shown to be above 60%, which makes the method suitable for qualitative characterization studies as well as quantitative studies in biomarker discovery.

Characterization of Short Peptides from Urine

For the investigation of native peptides from urine samples, an orthogonal separation strategy was adopted, in order to provide a global profile of urinary peptides. Conventional separation on C18 was complemented with data from HILIC separation on the iHILIC Fusion column. The iHILIC Fusion stationary phase is a modern generation zwitterionic phase, whose separation principle combines hydrophilic partitioning, weak electrostatic interactions, and hydrogen bonding. The iHILIC-Fusion is a charge-modulated hydroxyethyl amide HILIC, in which the cationic ammonium site is at the terminal position separated from mixed sulfate and phosphate anionic sites by a linker with hydroxyethyl amide side chains. The column has been employed for separation of two hallucinogenic alkaloids47, three dithiocarbamate fungicides48, glutamate, glutamine, and glufosinate49 in target analysis and for lipidomics50,51 and metabolomics in untargeted analysis52,53. In one of these works54, the iHILIC Fusion column provided better results than other HILIC columns, showing the largest number of detected standards and metabolite features when operated in acidic pH, with a good coverage of metabolite classes like amino acids. Given the potential for



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separation of short peptides, the iHILIC column was chosen for an orthogonal separation of peptide samples to the conventional C18. Extracted-ion chromatograms of the urine samples separated by C18 and HILIC are displayed in Figure 1a and b, respectively. The stability of the iHILIC was also evaluated over the nine technical replicates from the enriched urine samples. The separation proved robust, not only for reproducibility of the retention time of the identified peptides (Figure S4a) but also for the related area, which showed limited variations over the tested conditions. In particular, a boxplot was made using the log2(area) for each run, which indicated only minor variation over time, as the average log2(area) was consistent across the nine runs at ca. 25 (Figure S4b). The reproducibility was also evaluated by Pearson correlation, by plotting each run vs all the others, and again good results were obtained, with coefficients in the range 0.972-0.999 (Figure S4c).


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Figure 1. Extracted-ion chromatograms showing the separation of native short peptides identified in urine samples by C18 (a) or iHILIC® Fusion (b).

The results confirmed the expectations: by grouping the results from the 18 technical replicates (9 for each type of chromatographic separation), a total of 152 short peptides was overall identified from the enriched urine samples, divided into 101 identifications from the 9 C18 runs and 111 identifications from the 9 HILIC runs, with only 60 identifications (which correspond to 39%) in common between the two separation strategies (Figure 2, Table S4 for the complete list of peptide identifications). The orthogonality is reflected in the GRAVY distribution of the identified peptides as well, with the C18 unique peptides being close to 0 value, common peptides in between with an average GRAVY of -0.99 and HILIC unique peptides with an average GRAVY value of -1.78 (Figure 2).

Figure 2. Boxplot with the GRAVY index distribution of the peptides identified from urine samples enriched by GCB and detected by RP alone (C18), HILIC alone (HILC) or common to both chromatographic separations (Common).



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The enrichment is particularly relevant if compared to the dilute and shoot approach, which is the most widely used sample preparation in metabolomics approaches. The direct analysis of urine 1:1 diluted in mobile phase and analyzed by RP UHPLC allowed to identify only 25 short peptides sequences, all of them also detected in the GCB enriched samples. Though not assessed by quantitative targeted analysis, a theoretical 20-fold enrichment by the GCB procedure over the dilute and shoot procedure is effective in reducing suppression and in pre-concentrating low abundant short peptides in urine. Finally, the effect of collection time and protease inhibitors was evaluated. To evaluate the effect of time collection, the urine samples were left to stand at room temperature for 2 h before acidification and GCB clean-up. To evaluate the effect of proteases and artefacts formation, protease inhibitors were immediately added to fresh urine samples, which were then processed as described in the experimental section. The results from these experiments indicated that neither time nor inhibitors had an effect on the short peptides identified after the GCB clean-up. This is particularly relevant for a possible application to clinical research: the short native peptides in urine appear to be quite stable to collection time or storage and no production of artefact short peptides was observed due to residual enzymatic activity.

Conclusions

The paper describes the development of a clean-up and enrichment method specific for short peptides (di-, tri- and tetrapeptides) native in urine without derivatization. The short peptides were isolated from urine using the GCB Carbograph 4 sorbent, as it allows retention also of hydrophilic compounds by a complex interaction mechanism mostly based on RP interactions but also on polar interactions, especially with positively charged species. The method, developed on standards, was finally applied to urine and allowed clean-up and pre-concentration of short native peptides with a


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relevant improvement in total short peptide identifications compared to a common dilute and shoot approach, which is the most exploited one for metabolite analysis in urine. The use of orthogonal UHPLC separation by both RP and HILIC allowed to increase the total number of peptides, as the two separation strategies are complementary to each other. The HRMS method, based on data dependent acquisition mode, together with a semi-automated workflow for peptide MS and MS/MS spectra investigation, allowed the identification of 152 short peptides. The work provides a useful strategy for characterization of the short peptides which cannot be investigated by ordinary peptidomics protocols based on proteomics technologies. Compared to methods based on derivatization, the described approach does not require any sample pretreatment other than the SPE on GCB, which rules out possible analyte loss due to low reaction yields. Another advantage of the direct analysis of native peptides is the possibility of exploiting software for peptide in-silico fragmentation, which simplifies the matching procedure of MS/MS spectra and can eventually be automated for MS/MS matching and filtering of only the best candidate ions for manual validation; the use of in-silico fragmentation software also provides another advantage for the direct analysis, which is the possibility of suspect screening data analysis, which in turn allows to bypass the limitations of targeted analysis in complex matrices and virtually gives access to any potential short peptide sequence independent of the availability of pure standards. The developed method appears promising for the specific investigation of short peptides in urine as possible biomarkers of disease, filling an existing gap for this specific class of analytes. Nevertheless, efforts are still needed, namely to include important peptides with modifications and non-proteinogenic amino acids as building blocks of short peptides, which have not been considered in this study and which may potentially bear an important biological information. At the same time, the GCB enrichment procedure may be extended to other biofluids, in particular an application to plasma would be promising for better investigation of circulating native short peptides, also as possible biomarkers of a much larger range of diseases.



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In the regard of application of the developed method to clinical use, it is important to clearly state that the developed procedure, while providing a large coverage of short peptides in a complex matrix, still it is not sufficiently easy or automatable for routine clinical use, but could still be employed for discovery purposes. Method standardization, already described for clinical metabolomics and lipidomics55, would prove useful in the case the described method were used for biomarker discovery, as quality control samples need to be employed to ensure differences between samples are significant. Quality control should include not only pooled samples for HPLC-MS/MS check, but also a procedural control as well, processed in parallel to samples, to guarantee the reproducibility of sample preparation. As peptides can be only tentatively identified by this suspect screening approach, after statistical analysis of the acquired data, the candidate biomarker peptides need to be synthetized to confirm the identity by checking retention time and fragmentation pattern. Finally, a dedicated target method needs to be developed to quantitate the putative biomarkers in a large cohort study, for confirmation of the biological significance in disease.

Supporting Information

List of peptide standards with related mass, GRAVY value, molecular weight, pI and MRM acquisition conditions, optimization of GCB extraction on standard peptides, equations for calculation of recovery, matrix effect and process efficiency, chromatograms, scatter plots and boxplots for HILIC separation of peptides in urine (Supporting information), list of identified short peptides in urine (Table S4).

Acknowledgements


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The work was supported by the PRIN project Prot. 2017Y2PAB8, entitled “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”, provided by the Italian Ministry of Education, Universities and Research.

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