Detection of Podocin in Human Urine Sediment Samples by Charge Derivatization and LC-MS-MRM Method

International Journal of Molecular Sciences

Article Detection of Podocin in Human Urine Sediment Samples by Charge Derivatization and LC-MS-MRM Method

Remigiusz B ˛achor 1,* , Dorota G ˛aszczyk 1, Karolina Panek-Laszczy ´nska 2, Andrzej Konieczny 3 , Wojciech Witkiewicz 4, Piotr Stefanowicz 1 and Zbigniew Szewczuk 1

1 Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland; [email protected] (D.G.); [email protected] (P.S.); [email protected] (Z.S.) 2 1st Department and Clinic of Gynecology and Obstetrics, Wroclaw Medical University, 50-368 Wroclaw, Poland; [email protected] 3 Department of Nephrology and Transplantation Medicine, Wroclaw Medical University, 50-556 Wroclaw, Poland; [email protected] 4 Research and Development Center, Regional Specialized Hospital, 51-124 Wroclaw, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-71-375-7218; Fax: +48-71-328-2348

Received: 24 March 2020; Accepted: 30 April 2020; Published: 2 May 2020

Abstract: Detection of podocytes in urine might serve as a useful diagnostic tool in both primary and secondary glomerular diseases. The utility of podocyturia has been confirmed for both pre-eclampsia and glomerulonephritis. Here, we present a new and sensitive method for qualitative LC-MS-multiple-reaction-monitoring (MRM) analysis of podocin, serving as a podocyturia biomarker in urine sediments. The following podocin tryptic peptides with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223, 240SIAQDAK246, and 292MIAAEAEK299 sequences were applied as a model. The selective chemical derivatization of the ε amino group of C-terminal lysine residue in tryptic peptides, by 2,4,6-triphenylpyrylium salt (TPP) as a fixed charge tag, was employed to increase the ionization efficiency, in routine ESI-MS analysis. Additionally, the generation of a reporter ion, in the form of a protonated 2,4,6-triphenylpyridinium cation, makes the derivatized peptide analysis in the MRM mode unambiguous. Identification of derivatized and non-derivatized peptides were performed, and the obtained results suggest that the peptide with the 292MIAAEAEK299 sequence may serve as a marker of podocyturia.

Keywords: podocin; podocyturia; preeclampsia; charge derivatization; LC-MS; MRM

1. Introduction Podocyturia refers to the presence of podocytes, highly differentiated glomerular epithelial cells, maintaining the filtration barrier located in the outer space of the glomerular basement membrane (GBM) in urinary sediment [1]. Podocytes detected in urine are viable despite the lack of hostile glomerular environment and urinary acidity. Podocyturia may precede proteinuria, the traditional marker of glomerular injury, and reflect the disease activity. Therefore, in diseases affecting glomeruli, the presence and the quantification of urinary podocytes may be of remarkable relevance as it can herald the appearance of proteinuria. Preeclampsia (PE) is a severe pregnancy-related disorder affecting a large number of pregnant women, with delivery as the only cure [2–4]. Ten million women around the world develop PE each year and 76,000 of them die each year. The number of newborns dying because of this disorder is

Int. J. Mol. Sci. 2020, 21, 3225; doi:10.3390/ijms21093225 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3225 2 of 13 estimated at 500,000 per annum [5]. Symptoms of PE are defined as a presence of systolic blood pressure 140 mmHg and/or diastolic blood pressure 90 mmHg, as measured twice at least 6 h and less than ≥ ≥ 7 days apart, with the coexistence with one or more of the following new-onset conditions: Proteinuria, renal insufficiency, liver involvement, neurological complications, hematological complications, or uteroplacental dysfunction (fetal growth restriction) [6,7]. Although the absence of proteinuria does not exclude the appearance of PE, glomerular injury still plays a crucial role in its pathogenesis. The presence of podocytes, as well as podocyte-specific proteins (such as nephrin, synaptopodin, and podocin), in urine of pregnant women, suggests a possible appearance of PE at an early stage [8,9]. Currently, podocyte detection is a potential and promising candidate for being applied as a marker for diagnosis, treatment, and prevention of PE’s long-term complications [10]. It was also demonstrated that both nephrin and synaptopodin expression were reduced in contrast to the intact expression of podocin in women affected by PE [11]. Podocin has been determined as the most robust podocyte marker, compared to nephrin and synaptopodin, with 100% sensitivity in women with PE. Therefore, podocin is considered as one of the most podocyte-specific biomarkers of PE. Previously, we published a review describing identification methods of podocyte injury biomarkers in urine [12]. They involve a cell culture, accompanied with immunofluorescence, a quantitative polymerase chain reaction, and other immunoanalytical-based methodologies such as ELISA. However, all of the presented methods have some significant drawbacks such as the generation of antibodies [9]. In 2013, Garovic et al. [13] presented the application of mass spectrometry as a new diagnostic tool of podocyturia detection, in urine samples, among women with diagnosed PE. In this method, the pellet was digested by trypsin and the obtained hydrolysates were analyzed by the LC-MS-multiple-reaction-monitoring (MRM) technique, in the presence of isotopically labeled internal standard. The podocin tryptic fragment, with the 39QEAGPEPSGSGR50 sequence, was described as the most promising for podocin quantification. Additionally, achieved results were confirmed by ELISA tests. The analysis was performed using 50 mL of urine sample. Another approach was presented by Simon et al. [14]. In their study, podocin was quantified in the soluble fraction of urine. The tryptic podocin peptide, with the 59APAATVVDVDEVR71 sequence, was chosen for podocin quantification by LC-MS-MRM. The described results clearly confirmed the usefulness of the LC-MS-MRM method as a diagnostic tool for podocin detection in urine. The ESI-MS analysis of tryptic peptides, containing lysine residue at the C-terminus, may be difficult for MS analysis due to their low ionization efficiency. Therefore, increasing the sensitivity of this method may allow analysis of trace amounts of the biomarkers in the early stage of preeclampsia. Additionally, Martineau and co-workers [15] used the ultra-performance liquid chromatography tandem mass spectrometry technique for simultaneous quantitation of podocalyxin and podocin in urine specimens. We also applied the LC-MS-MRM technique, in analysis of podocin, in tryptic digests of feline [16] and canine [17] urine samples. The evaluated group of animals consisted of cats and dogs with diagnosed chronic kidney disease. The performed study has proved that the proposed method may be applied in the analysis of podocin in animal urine samples. Based on previous results, further research was conducted, using horse urine samples, because there are no biomarkers in this species specific for diagnosing acute kidney injury (AKI) in the asymptomatic stage. However, to increase the sensitivity, the proposed method needs further optimization. Recently, we developed a method of increasing the sensitivity of tryptic peptides by mass spectrometry analysis, by their derivatization with ionization enhancers, containing the quaternary ammonium group and their analysis by tandem mass electrospray mass spectrometry, hyphenated with liquid chromatography (LC-ESI-MS/MS) [18]. The fixed charge tag proposed by us, in the form of 2,4,6-triphenylpyrylium salt, in the reaction with the ε-amino group of lysine residue, resulted in the formation of the 2,4,6-triphenylpyridinium derivative [19,20]. Such a charge tag increases the ionization efficiency during mass spectrometry analysis, making possible the sensitive analysis of trace numbers of compounds. The application of inexpensive and commercially available pyrilium salt as a derivatization reagent allows selective derivatization of the ε-amino group of lysine, containing Int. J. Mol. Sci. 2020, 21, 3225 3 of 13 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 13 peptidescontaining present peptides in complex present mixtures. in complex Peptides modifiedmixtures. by thePeptides 2,4,6-triphenylpyridinium modified by the group 2,4,6- in MStriphenylpyridinium/MS experiments generate group the in protonated MS/MS 2,4,6-triphenylpyridinium experiments generate ion, the serving protonated as a reporter 2,4,6- ion intriphenylpyridinium multiple-reaction-monitoring ion, serving (MRM) as a reporter mode. Moreover, ion in multiple-reaction-monitoring the developed derivatization (MRM) of peptides mode. is inexpensiveMoreover, the and developed easy to perform, derivatization even by of an peptides inexperienced is inexpensive analyst. and The 2,4,6-triphenylpyryliumeasy to perform, even by salt an wasinexperienced also applied analyst. for sensitive The 2,4,6-triphenylpyrylium ESI-MS detection of cysteine-containing salt was also applied peptide, for captured sensitive by properly ESI-MS designeddetection solidof cysteine-containing support [21]. peptide, captured by properly designed solid support [21]. InIn thisthis manuscript, manuscript, we proposewe prop aose new a and new sensitive and sensitive method of method tryptic podocinof tryptic peptide podocin identification peptide inidentification urine sediments, in urine based sediments, on the based sample on derivatizationthe sample derivatization by a fixed by charge a fixed tag, charge in the tag, form in the of 2,4,6-triphenylpyryliumform of 2,4,6-triphenylpyrylium salt and thesalt targetingand the targeting LC-MS-MRM LC-MS-MRM technique. technique.

2.2. Results TheThe aimaim ofof thisthis workwork waswas toto investigateinvestigate thethe applicabilityapplicability ofof podocinpodocin tryptictryptic peptidepeptide chargecharge derivatization,derivatization, by 2,4,6-triphenylpyridinium2,4,6-triphenylpyridinium salt, to increase their ionizationionization eefficiency,ﬃciency, allowingallowing ultrasensitiveultrasensitive detectiondetection byby thethe LC-MS-MRMLC-MS-MRM method.method. The tryptictryptic humanhuman podocinpodocin peptidespeptides withwith thethe 169169LQTLEIPFHEIVTK182182, ,213213AVQFLVQTTMKAVQFLVQTTMK223223, 240, 240SIAQDAKSIAQDAK246, 246and, and 292MIAAEAEK292MIAAEAEK299 sequences299 sequences were werechosen chosen as potential as potential podocin podocin biomarkers biomarkers (Figure (Figure 1)1. ).According According to to the the Protein Protein Information ResourceResource DatabaseDatabase (https:(https://proteininform//proteininformationresource.orgationresource.org/pir/pirwwwwww/index.shtml),/index.shtml), all ofall the of peptides the peptides are present are onlypresent in human only in podocin.human podocin.

. Figure 1. Sequence of human podocin with marked trypsin cleavage sites determined according Figure 1. Sequence of human podocin with marked trypsin cleavage sites determined according to to the UniProt databases (entry name PODO_HUMAN). The selected sequences were underlined. the UniProt databases (entry name PODO_HUMAN). The selected sequences were underlined. Arrow indicates the trypsinolysis site. Arrow indicates the trypsinolysis site. First, we determined the detection sensitivity of model synthetic peptides, using LC-MS/MS methods.First, Thewe determined data revealed the thatdetection the 169 sensitivitH-LQTLEIPFHEIVTK-OHy of model synthetic182 peptides,peptide mayusing be LC-MS/MS analyzed methods. The data revealed that the 169H-LQTLEIPFHEIVTK-OH16 182 peptide may be analyzed even at even at the subfemtomolar level of detection (6.0 10− mole). Other peptides were identiﬁed the subfemtomolar15 level292 of detection 299(6.0 × 10−16 mole).15× Other 240peptides were246 identified at 1.215 × 10−15 at 1.2 10− mole for MIAAEAEK , 6.4 10− mole for SIAQDAK , and 7.9 10− mole × 292 299 −15 × 240 246 −×15 formole213 AVQFLVQTTMKfor MIAAEAEK223., The 6.4 performed× 10 mole experiment for SIAQDAK allowed peptides, and to7.9 be × chosen10 mole with thefor 213 223 169 169AVQFLVQTTMKH-LQTLEIPFHEIVTK-OH. The 182performedsequence experiment as a potential allowed model forpeptides sensitive to detection be chosen and with identiﬁcation the H- 182 ofLQTLEIPFHEIVTK-OH podocin in human urine sequence sediment as a samples. potentialFor model each for of se thensitive synthesized detection peptides,and identification the MRM of podocin in human urine sediment samples. For each of the synthesized peptides, the MRM methods were optimized and the following transitions were applied, for podocin investigation: For

Int. J. Mol. Sci. 2020, 21, 3225 4 of 13

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 13 methods were optimized and the following transitions were applied, for podocin investigation: 169 182 2+ For169LQTLEIPFHEIVTKLQTLEIPFHEIVTK182 [M +[M 2H]+2+2H] (m/z 834.47)(m/z 834.47) ion, as ion, the as most the mostintensive intensive signal signal on the on obtained the obtained mass 213 223 2+ mass spectra, 834.47 242.10 (b ), 834.47 970.60 (y );213 for AVQFLVQTTMK223 [M + 2+2H] (m/z 633.35) spectra, 834.47→242.10→ (b2), 834.472 →970.60→ (y8); for8 AVQFLVQTTMK [M + 2H] (m/z 633.35) ion, ion,as the as themost most intensive intensive signal signal on the on theobtained obtained mass mass spectra, spectra, 633.35 633.35→72.1072.10 (b1), (b 633.351), 633.35→143.05143.05 (a2); (a for2); 240 246 292 → 299 → for240 SIAQDAK246 732.40 218.16 (y ), 732.40 461.26 (y ); for 292 MIAAEAEK299 862.45 347.20 (y ), SIAQDAK 732.40→218.16→ (y2), 2732.40→→461.26 (y4);4 for MIAAEAEK 862.45→347.20 (y33), 862.45 547.28 (y ). The urine sediment samples were prepared according to the procedure described 862.45→→547.28 (y55). The urine sediment samples were prepared according to the procedure described inin the the Section materials4. The and achieved methods results section. for The the sampleachieved of healthyresults for subjects the sample (black line)of healthy and with subjects diagnosed (black PEline) (red and line) with are diagnosed presented PE below (red (Figureline) are2). presented below (Figure 2).

FigureFigure 2.2. Multiple-reaction-monitoringMultiple-reaction-monitoring(MRM) (MRM)analysis analysisof ofurine urine sedimentsediment tryptictryptic digestdigest fromfrom healthyhealthy subjectssubjects (black (black line) line) and and pregnant pregnant women women with with diagnosed diagnosed preeclampsia preeclampsia (red (red line). line).

InIn thethe casecase ofof healthyhealthy individuals,individuals, signalssignals correspondingcorresponding toto thethe investigatedinvestigated peptidespeptides areare absentabsent (Figure(Figure2 ,2, black black line). line). It It was was found found that that urinary urinary podocyte podocyte excretion excretion may may occur occur in in the the case case of of healthy healthy patientspatients ( (<0.5<0.5 podocytespodocytes/mg/mg creatinine)creatinine) [[22];22]; however,however, thethe numbernumber ofof releasedreleased cellscells andand podocinpodocin maymay notnot be be su sufficientfficient for for the the identification identification by commonly by commo usednly analyticalused analytical techniques. techniques. Garovic Garovic and co-workers and co- claimedworkers [claimed23] that podocytes[23] that podocytes were absent were in absent urine in among urine among healthy healthy pregnant pregnant women women and those and withthose gestationalwith gestational hypertension hypertension but without but without other symptomsother symptoms of PE. of PE. TheThe presented presented MRM MRM chromatograms chromatograms for allfor of all the of selected the selected and synthesized and synthesized tryptic human tryptic podocin human 292 299 peptidespodocin showpeptides only signalsshow only corresponding signals correspondi to MRM transitionsng to MRM of peptide, transitions with theof peptide,MIAAEAEK with the sequence,292MIAAEAEK in the299 urine sequence, sediment in the sample urine from sediment pregnant sample woman from with pregnant diagnosed woman PE (Figurewith diagnosed2, red line). PE Signals(Figure corresponding2, red line). Signals to the corresponding fragment ion to of the other fragment analyzed ion of peptides other anal haveyzed not peptides been identified. have not Therebeen areidentified. two possible There explanationsare two poss ofible such explanations an observation. of such First, an the observation. tryptic digestion First, the of humantryptic podocindigestion may of human result in podocin the formation may result of a smallin the number formation of these of a small peptides number or have of these not even peptides been formedor have atnot all. even Another been formed possibility at all. is Another that podocin possibility may existis that in podocin two isoforms may exist – canonical in two isoforms and an ill-defined– canonical shortand an one. ill-defined A shorter short 315 aminoone. A acid shorter isoform 315 ofamin humano acid podocin isoform lacks of human one exon podocin encoding lacks the one central exon partencoding of the the prohibition central part domain of the (PHB-domain) prohibition domain [24,25]. (PHB-domain) The presence of[24,25]. a short The podocin presence isoform of a short was podocin isoform was determined by Volker and co-workers using mass spectrometry [26]. The role of the short isoform of podocin is still unknown and may differ from the canonical variant. It was suggested that the short isoform may affect the lipid and protein composition of the slit diaphragm.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 13

Based on the sequence of the short human podocin isoform (UniProt identifier: Q9NP85-2), in the missing fragment, with the 180VTKDMFIMEIDAICYYRMENASLLLSSLAHVSKAVQFLVQTTMKRLLAHRSLTEILLERKSIAQD AKV247 sequence, the chosen model peptides with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223 Int.and J. Mol.240SIAQDAK Sci. 2020, 21246, 3225 sequences were included. Additionally, according to the Protein Information5 of 13 Resource Database, sequences presented above are characteristic only for the canonical podocin isoform, whereas the 292MIAAEAEK299 sequence is characteristic for both the canonical and short determinedpodocin isoform. by Volker Therefore, and co-workers whether the using short mass isofor spectrometrym of podocin [26 ].is Thepresent role in of PE, the the short identification isoform of podocinof the presented is still unknown model peptides and may is di ffnoter frompossible. the canonicalHowever, variant. there are It wasno literature suggested data that describing the short isoformwhich podocin may affect isoform the lipid is more and protein characteristic composition for podocyturia. of the slit diaphragm. Additionally, Based both on Garovic the sequence and co- of theworkers short human[11] and podocin Simon et isoform al. [14] (UniProt described identifier: the tryptic Q9NP85-2), human podocin in the missing peptide fragment, sequences, with which the 180 247 mayVTKDMFIMEIDAICYYRMENASLLLSSLAHVSKAVQFLVQTTMKRLLAHRSLTEILLERKSIAQDAKV be used as a podocin marker, based on the sequence and trypsynolysis of the short podocin 169 182 213 223 sequenceisoform., Therefore, the chosen in model our work, peptides the applicability with the LQTLEIPFHEIVTKof other tryptic podocin, fragments,AVQFLVQTTMK originating fromand 240 246 theSIAQDAK canonical form,sequences as a potential were included. podocin Additionally, marker, which according might tobe theidentified Protein by Information LC-MS-MRM, Resource was Database,the main goal. sequences presented above are characteristic only for the canonical podocin isoform, 292 299 whereasAccording the MIAAEAEK to the peptide selectionsequence criteria is characteristic for proteomic for analysis, both the presented canonical by and Mohammed short podocin and isoform.co-workers Therefore, [27], methionine whether theresidue short should isoform be of abse podocinnt in the is presentpeptide in of PE, interest the identification due to the possible of the presentedoxidation modelto sulfoxide peptides (+16 is Da not mass possible. shift), However,which may there reduce are the no sensitivity literature dataduring describing MRM analysis. which podocinTo check isoform the stability is more characteristicof the selected for podocyturia.peptide, with Additionally, the 292MIAAEAEK both Garovic299 sequence, and co-workers under [11the] andconditions Simon etapplied al. [14] during described sample the tryptic preparation, human podocinthe 0.1 mg peptide of this sequences, peptide was which added may beto usedthe urine as a podocinsediment marker, sample. based After on all the steps, sequence the LC-MS and trypsynolysisanalysis was performed of the short in podocin the selected isoform. ion monitoring Therefore, inmode our work,to analyze the applicability the possibility of other of methionine tryptic podocin oxidation. fragments, The m/z originating values for from the thenative canonical (862.45 form, [M + asH] a+) potential and oxidized podocin form marker, (878.43 which [M + H] might+) were be identifiedmonitored by (Figure LC-MS-MRM, 3A,B). The was obtained the main results goal. clearly confirmedAccording that tounder the peptidethe applied selection conditions, criteria the for model proteomic peptide analysis, did not presented undergo by oxidation. Mohammed and co-workersAdditionally, [27], methionine the obtained residue sample should was stored be absent at room in the temperature peptide of for interest one week, due to and the even possible after oxidationthis time, tomethionine sulfoxide (oxidation+16 Da mass was shift), not observed. which may To reduce clearly the demonstrate sensitivity the during lack MRM of methionine analysis. 292 299 Tooxidation, check the under stability the of applied the selected conditions peptide, during with the the sampleMIAAEAEK preparation,sequence, we obtained under the the conditions sulfoxide appliedderivative during of samplethe model preparation, peptide, the developed 0.1 mg of thisthe peptideMRM method, was added and to thethe urine following sediment transitions sample. After all steps, the LC-MS analysis was performed in the selected ion monitoring mode to analyze the 878.43→532.24 (b5) and 878.43→261.13 (b2) were monitored in the urine sample, in which the native + possibilityform of the of MIAAEAEK methionine oxidation.peptide was The identifiedm/z values (Figur for thee 3C–F). native The (862.45 achieved [M + resultsH] ) and clearly oxidized confirmed form + (878.43the lack [M of+ oxidation.H] ) were Therefore, monitored it (Figure may be3A,B). conclu Theded obtained that the results proposed clearly peptide confirmed may that be used under in the the appliedpodocin conditions, investigation the in model urine peptide sediment did samples. not undergo oxidation.

A) M(O)IAAEAEK 878.43

250

1500 B) MIAAEAEK 862.45 1000

500 0

C) M(O)IAAEAEK 878.43→532.24 (b ) 200 5

100

75 D) M(O)IAAEAEK 878.43→261.13 (b2)

INTENSITY 50

25 0

2000 E) MIAAEAEK 862.45→347.20 (y3)

1000

1500 F) MIAAEAEK 862.45→547.28 (y5) 1000

500 0

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min RETENTION TIME [min] Figure 3. Selected ion monitoring of M(O)IAAEAEK (A) and MIAAEAEK (B) peptide sequences in urine sample tryptic digest from patient with diagnosed preeclampsia (PE). MRM investigation of M(O)IAAEAEK (C,D) and MIAAEAEK (E,F) peptide sequences in urine sediment tryptic digest from pregnant women with diagnosed preeclampsia. MRM transitions corresponding to the oxidized peptide with the M(O)IAAEAEK sequence were not identiﬁed. Int. J. Mol. Sci. 2020, 21, 3225 6 of 13

Additionally, the obtained sample was stored at room temperature for one week, and even after this time, methionineInt. J. Mol. Sci. oxidation 2020, 21, x FOR was PEER not observed. REVIEW To clearly demonstrate the lack of methionine oxidation, 6 of 13 under the applied conditions during the sample preparation, we obtained the sulfoxide derivative of the model peptide,Figure 3. developed Selected ion the monitori MRMng method, of M(O)IAAEAEK and the following (A) and transitions MIAAEAEK 878.43 (B) peptide532.24 sequences (b ) and in → 5 878.43 261.13urine (b sample) were tryptic monitored digest infrom the patient urine sample,with diagnosed in which preeclampsia the native form(PE). ofMRM the investigation MIAAEAEK of → 2 peptide wasM(O)IAAEAEK identified (Figure (C,D)3 andC–F). MIAAEAEK The achieved (E,F) peptide results sequences clearly confirmed in urine sediment the lack tryptic of oxidation. digest from Therefore, itpregnant may be concludedwomen with that diagnosed the proposed preeclampsia. peptide MR mayM betransitions used in thecorresponding podocin investigation to the oxidized in urine sedimentpeptide samples. with the M(O)IAAEAEK sequence were not identified. Due to the fact that in the performed analysis, only the peptide with the 292MIAAEAEK299 292 299 sequence wasDue identified to the fact in thethat urine in the sediment perform sample,ed analysis, from women only the with peptide PE, we with decided the toMIAAEAEK analyze the presencesequence of thiswas trypticidentified podocin in the fragment urine sediment by LC-MS-MRM sample, from in thewomen urine with sediment PE, we samples decided from to analyze patientsthe with presence diagnosed of this membranous tryptic podocin nephropathy fragment (MN), by LC-MS-MRM focal segmental in the glomerulosclerosis urine sediment samples (FSGS), from membranoproliferativepatients with diagnosed glomerulonephritis membranous (MPGN), nephropa andthy IgA (MN), nephropathy focal segmental (IgAN). Theglomerulosclerosis obtained results(FSGS), are presented membranoproliferative in Figure4A–H. glomerulonephritis (MPGN), and IgA nephropathy (IgAN). The obtained results are presented in Figure 4 (panels A–H).

Figure 4. MRM investigation of 292MIAAEAEK299 peptide in urine sediment tryptic digest from patient Figure 4. MRM investigation of 292MIAAEAEK299 peptide in urine sediment tryptic digest from patient with diagnosed membranous nephropathy (MN) (A,B); focal segmental glomerulosclerosis (FSGS) with diagnosed membranous nephropathy (MN) (A,B); focal segmental glomerulosclerosis (FSGS) (C,D); membranoproliferative glomerulonephritis (MPGN) (E,F); and IgA nephropathy (G,H). (C,D); membranoproliferative glomerulonephritis (MPGN) (E,F); and IgA nephropathy (G,H). The signals corresponding to the MRM transition of fragment ions of the 292MIAAEAEK299 292 299 peptide areThe present signals in panels corresponding A,B and G,H,to the obtained MRM transition after tryptic of digestfragment analysis ions ofof urinethe sedimentMIAAEAEK samplespeptide from patientsare present with in diagnosed panels A,B MN and (Figure G,H, obtained4A,B) and after IgAN tryptic (Figure digest4G,H). analysis On other of urine MRM sediment chromatograms,samples from transitions patients corresponding with diagnosed to theMN analyzed (Figure 4, peptide panels were A,B) notand identified IgAN (Figure (Figure 4, 4panelsC–F). G,H). AnalysisOn other of aMRM small chromatograms, number of peptides transitions by ESI-MS corr mayesponding be limited to the due analyzed to the low peptide ionization were not efficiencyidentified of analytes. (Figure Therefore, 4, panels to C–F). overcome this problem, ionization enhancers are commonly used. We decidedAnalysis to apply of a strategy,a small developednumber of bypeptides us, of peptideby ESI-MS modification may be limited by 2,4,6-triphenylpyrilium due to the low ionization salt asefficiency an ionization of analytes. marker Therefore, [15,28]. The to overcome model peptide this problem, with the ionization292MIAAEAEK enhancers299 sequence are commonly was used. We decided to apply a strategy, developed by us, of peptide modification by 2,4,6-triphenylpyrilium salt as an ionization marker [15,28]. The model peptide with the 292MIAAEAEK299 sequence was modified according to the procedure described in the materials and methods section. The obtained H-MIAAEAEK(TPP+)-OH derivative was used for MRM method development. For the selected 2,4,6- triphenylpyrylium salt (TPP)-peptide derivative, the MRM method was optimized using the [M +

Int. J. Mol. Sci. 2020, 21, 3225 7 of 13 modiﬁed according to the procedure described in the Section4. The obtained H-MIAAEAEK(TPP +)-OH Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 13 derivative was used for MRM method development. For the selected 2,4,6-triphenylpyrylium salt 3+ (TPP)-peptide2H]3+ (m/z 653.1) derivative, ion as the the most MRM intensive method signal was optimized on the obtained using the mass [M +spectra.2H] (Them/z 653.1)following ion asMRM the most intensive signal on the obtained mass spectra. The following+ MRM transitions2+ 576.78 637.30 transitions 576.78→637.30 (y3) and 576.78→308.20 ([TPP + H] ) from the [M + H] ion were→ selected (y ) and 576.78 308.20 ([TPP + H]+) from the [M + H]2+ ion were selected and used in the analysis and3 used in the→ analysis of the presence of human podocin in the urine sediment samples. The ofachieved the presence results offor human the analyzed podocin samples in the originatin urine sedimentg from healthy samples. subjects The achieved (Figure 5, results panels for A,B), the a analyzedpatient with samples diagnosed originating PE (Figure from 5, healthy panels C,D) subjects, membranous (Figure5A,B), nephropathy a patient (Figure with diagnosed 5, panels E,F), PE (FigureFSGS (Figure5C,D), membranous5, panels G,H), nephropathy MPGN (Figure (Figure 5, 5panelsE,F), FSGS I,J), and (Figure IgAN5G,H), (Figure MPGN 5, panels (Figure K,L)5I,J), are andpresented IgAN (Figurebelow. 5K,L) are presented below.

100 A) MIAAEAEK(TPP) 576.78→637.30 (y3)

150 B) MIAAEAEK(TPP) 576.78→308.20 [TPP+H]+ 100

C) MIAAEAEK(TPP) 576.78→637.30 (y ) 2000 3

1000

7500 D) MIAAEAEK(TPP) 576.78→308.20 [TPP+H]+

5000

2500

E) MIAAEAEK(TPP) 576.78→637.30 (y3) 2500

5000 F) MIAAEAEK(TPP) 576.78→308.20 [TPP+H]+

2500

200 G) MIAAEAEK(TPP) 576.78→637.30 (y3) INTENSITY

100

0 300 H) MIAAEAEK(TPP) 576.78→308.20 [TPP+H]+

150

200 I) MIAAEAEK(TPP) 576.78→637.30 (y3)

100

200 J) MIAAEAEK(TPP) 576.78→308.20 [TPP+H]+

100

2000 K) MIAAEAEK(TPP) 576.78→637.30 (y3)

1000

3000 L) MIAAEAEK(TPP) 576.78→308.20 [TPP+H]+ 2000

1000

0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min RETENTION TIME [min] FigureFigure 5.5. MRMMRM analysisanalysis ofof MIAAEAEK(2,4,6-triphenylpyryliumMIAAEAEK(2,4,6-triphenylpyrylium saltsalt (TPP))(TPP)) peptidepeptide conjugateconjugate inin tryptictryptic digestdigest ofof urineurine sedimentsediment samplessamples obtainedobtained fromfrom healthyhealthy patientpatient ((AA,,BB),), patientpatient withwith PEPE ((CC,,DD),), MNMN ((EE,F,F),), FSGS FSGS ( G(G,H,H),), MPGN MPGN ( I(I,J,J),), and and IgA IgA nephropathy nephropathy (IgAN) (IgAN) ( K(K,L,L).).

TheThe obtained MRM MRM data data (Figure (Figure 5)5) for for the the ur urineine sediment sediment samples, samples, derivatized derivatized by 2,4,6- by 2,4,6-triphenylpyriliumtriphenylpyrilium salt saltas an as anionization ionization marker, marker, present present peaks peaks corresponding corresponding to to thethe transitiontransition characteristic for the selected human tryptic podocin peptide with the 292MIAAEAEK299 sequence, in the case of the urine sediment sample from pregnant women with PE (Figure 5, panels C,D), MN (Figure 5, panels E,F), and IgAN (Figure 5, panels K,L). The intensive MRM transitions were observed only in these cases, additionally confirmed by the retention time of the 292MIAAEAEK(TPP)299

Int. J. Mol. Sci. 2020, 21, 3225 8 of 13 characteristic for the selected human tryptic podocin peptide with the 292MIAAEAEK299 sequence, in the case of the urine sediment sample from pregnant women with PE (Figure5C,D), MN (Figure5E,F), and IgAN (Figure5K,L). The intensive MRM transitions were observed only in these cases, additionally confirmed by the retention time of the 292MIAAEAEK(TPP)299 conjugate. Moreover, the urine sediment sample from healthy individuals (Figure5A,B) showed a lack of analyzed tryptic podocin sequence. The same result was observed in the case of patients with diagnosed FSGS (Figure5G,H) and MPGN (Figure5I,J). The advantage of the applied pyrylium salt, as reagents for ionization, tagging of peptides lies in the high regioselectivity of this reagent – it reacts with sterically unhindered primary amino groups such as the ε-amino group of lysine residue, the fragmentation patterns of labeled peptides, and the possibility of the convenient and economic incorporation of the isotopic label. The 2,4,6-triphenylpyridinium-modified peptides generate an abundant protonated 2,4,6-triphenylpyridinium ion (m/z 308) in MS/MS experiments. This fragment serves as a reporter ion for the multiple-reaction-monitoring analysis. The intensive signal corresponding to the [TPP + H]+ ion was observed by us and is presented in Figure5. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the derivatization simplicity of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides, which we plan to present in our future manuscript. In our study, we analyzed the presence of podocin tryptic peptides, with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223, 240SIAQDAK246, and 292MIAAEAEK299 sequences, in urine sediment samples from healthy subjects and those with diagnosed preeclampsia, membranous nephropathy, focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, and IgA nephropathy. We analyzed more than ten samples from healthy pregnant women and other healthy subjects. In the case of subjects with renal dysfunctions, we analyzed five samples for each case. The data obtained after LC-MS-MRM analysis of urine sediment tryptic digests of subjects with diagnosed preeclampsia, membranous nephropathy, focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, and IgA nephropathy were always the same.

3. Discussion All analyzed samples concerned diseases associated with glomerular injury. Garovic and co-workers reported that the podocyte detection, in the urine of preeclamptic women, may serve as a diagnostic tool in PE due to its strong predictive value [19]. Additionally, authors found that podocin is absent in urine sediment derived from healthy pregnant women and those with gestational hypertension. Furthermore, Aita et al. reported higher urinary podocyte excretion in the third trimester of pregnancy in patients with PE [29]. Therefore, it may be concluded that the received results correlate with the observations presented in the literature. MN, a major cause of nephrotic syndrome in adults, is related to the accumulation of immune deposits on the outer aspect of the glomerular capillary wall. In renal biopsies of patients affected by MN, podocyte cell injury and rearrangements of the glomerular basement membrane, leading to proteinuria, were found [30]. The podocytes’ damage causes their detachment and, as a consequence, lead to their presence in urine, enabling their identification. In the case of the analyzed sample (Figure5E,F), the presence of a signal corresponding to the podocin tryptic peptide confirms the presence of podocin. IgAN (Berger’s disease) is most often the primary glomerular disease, related to immunoglobulin A deposition in the glomerular mesangium. Recent literature data suggest a connection between the increased severity of glomerular dysfunction and lower number of glomerular podocytes [31]. The determination of urine podocyte number may serve as a diagnostic tool to differentiate between glomerular and non-glomerular disease and between inflammatory and noninflammatory disease [32]. Our results confirmed such an observation (Figure5K,L). Int. J. Mol. Sci. 2020, 21, 3225 9 of 13

FSGS is another type of primary glomerulonephritis, presenting frequently in form of the nephrotic syndrome. Reidy and Kaskel proved the crucial role of podocyte loss in the FSGS [33]. It was found that podocytes’ damage initiates their detachment from the glomerular membrane [34,35]. It may be assumed that due to their excretion to urine, podocin and its tryptic fragments may be identified in the urine sediment samples [32]. By contrast, Agrawal and co-workers found the reduction of glomerular podocin expression in FSGS [36], which may result in the low level or even lack of this protein in urine. Based on our results, podocin was not observed in the urine sediment samples from patients with diagnosed FSGS. The possible explanation of this may be the fact that FSGS not only reflects the primary affection of glomeruli but might also be a common end-path for other primary glomerular diseases. Possibly, in this case, FSGS reflected to the inactive disease, where proteinuria was only the sign of residual scarring; therefore, we were not able to detect podocin. The same result was observed in the case of urine samples from the patient with diagnosed membranoproliferative glomerulonephritis. The lack of MRM transitions on the obtained chromatograms (Figure4E,F, Figure5I,J) confirmed the absence of podocin in the analyzed sample. MPGN is a kidney disorder characterized by mesangial cell proliferation and structural changes in glomerular capillary walls [37]. The reduction in glomerular podocin expression in MPGN was also presented [33]. It may also be concluded that in this case, the lack of podocin tryptic peptide, confirmed by the LC-MS-MRM method, in the analyzed urine sediment sample from patients with MPGN has confirmation in literature data.

4. Materials and Methods

4.1. Chemicals All chemicals were used as supplied. Fmoc amino acid and Fmoc-Lys(Mtt)-Wang resin (0.56 mmol/g) were purchased from Novabiochem (Darmstadt, Germany). N-[(Dimethylamino)-1H- 1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) and trifluoroacetic acid (TFA) were obtained from IrisBiotech. Solvents for peptide synthesis (N,N-dimethylformamide (DMF), dichloromethane (DCM), N-ethyldiisopropylamine (DIEA), tetraethylammonium bicarbonate (TEAB) and N,N,N-triethylamine, 1,2-dithiotreitol (DTT), H2O2, tetrafluoroborate 2,4,6-triphenylpyrylium salt, and iodoacetamide) were obtained from Sigma Aldrich (Saint Louis, MO, USA); triisopropylsilane (TIS) was purchased from Fluka (Bucharest, Romania). Amicon® Ultra Centrifugal Filters were purchased from Merck (Darmstadt, Germany), Rapi-Gest™ SF was from Waters (Waters Chromatography Europe BV, Etten-Leur, Netherlands).

4.2. Peptide Synthesis Model peptides were synthesized manually on Fmoc-Lys(Boc)-Wang resin in Intavis AG syringe reactors containing polyethylene ﬁlters, using the Fmoc (9-ﬂuorenylmethoxycarbonyl) solid-phase synthesis procedure [38].

4.3. In-Solution Peptide Derivatization with 2,4,6-Triphenypyrilium Salt The purified model peptides (0.2 mg) were dissolved (in a separate Eppendorf tube) in 0.5 mL of DMF containing 0.05 mg of 2,4,6-triphenylpyrilium tetrafluoroborate (TPP), and 19 µL of N,N,N-triethylamine was added. The mixture was incubated at 60 ◦C for 1 h and the solution was evaporated under a nitrogen stream. The final product was dissolved in 5% MeCN/H2O mixture (v:v), and lyophilized and used for MS/MS analysis and MRM method development.

4.4. Puriﬁcation The obtained peptides were puriﬁed on a HPLC Thermo Separation system with a YMC-Pack RP C18 column (4.6 250 mm, 5 µm), UV detection (210 nm). Elution gradients were 0%–40% B in × Int. J. Mol. Sci. 2020, 21, 3225 10 of 13

A (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile/H2O, 4:1) for 30 min (ﬂow rate 1 mL/min). The collected fractions lyophilized.

4.5. Mass Spectrometry ESI-MS experiments were performed on a micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany) with a standard ESI source. The instruments parameters were as follows: Positive-ion mode, calibration with the Tunemix™ mixture (Agilent Technologies, Palo Alto, CA, USA), mass accuracy was better than 5 ppm, scan range: 50–1600 m/z; drying gas: Nitrogen; flow rate: 4.0 L/min, temperature: 200 ◦C; potential between the spray needle and the orifice: 4.2 kV analyte (flow rate: 3 µL/min.

4.6. CID MS/MS experiments were performed for singly ([M + H]+) and doubly protonated ([M + 2H]2+) precursor ions. Collision gas – argon. The collision energy was optimized between 10 and 30 V to obtain the best quality of fragmentation. Bruker Compass DataAnalysis 4.0 software was used for data analysis.

4.7. Urine Sample Preparation Collected urine samples obtained from patients with diagnosed membranous nephropathy, focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy ( 1 mL each) were centrifuged (10 min at 5000 rpm, 30 C). The obtained pellet was used ∼ ◦ for further experiments. A solid fraction was re-suspended in the mixture of = 0.1 M TEAB (1 mL) buﬀer containing Rapi-Gest™ SF (0.1%) and sonicated for 5 min. The sample was then centrifuged in an Amicon® Ultra Centrifugal Filter (4000 rpm,15 min). The obtained mixture was diluted with 0.1 M TEAB (1 mL) buﬀer and centrifuged again. Then, the sample was treated by 100 µL of 0.2 M DTT and incubated for 1 h at 30 ◦C. After that, the mixture was centrifuged (4000 rpm for 15 min) to remove DTT. Then, 100 µL of 0.1 M iodoacetamide was added and incubated at room temperature for 1 h. Then, the sample was washed with 0.1 M TEAB and centrifuged to remove the excess of iodoacetamide. The obtained supernatant was transferred into the Eppendorf tube, the 50 µg of trypsin in 200 µL of 0.1 M TEAB was added, and the sample was incubated at 37 ◦C overnight. The enzymatic reaction was terminated by addition of 20 µL of formic acid; after lyophilization, the dry solid was dissolved in 5% MeCN/H2O and analyzed by LC-MS.

4.8. Urine Tryptic Digest Derivatization with 2,4,6-Triphenypyrilium Salt The lyophilized tryptic urine hydrolates were dissolved in 0.5 mL of DMF solution containing 0.25 mg of 2,4,6-triphenylpyrilium tetraﬂuoroborate and 95 µL of N,N,N-triethylamine. The mixture was incubated at 60 ◦C for 1 h and the solution was evaporated under the nitrogen stream. The ﬁnal product was dissolved in 5% MeCN/H2O mixture (v:v) and lyophilized.

4.9. H2O2 Oxidation of Model Peptide Hydrogen peroxide stock (5 µL) was spiked into a 195 µL solution containing 0.1 mg/mL MIAAEAEK peptide in a mildly acidic buﬀer. The samples were incubated at 37 ◦C for 2 h, quenched with DTT, dissolved with water (500 µL), and lyophilized.

4.10. Liquid Chromatography - Mass Spectrometry (LC-MS) Analysis in Multiple-Reaction-Monitoring (MRM) Mode LC-MS/MRM analysis was carried out on a LCMS-8050 Shimadzu apparatus, equipped with a UHPLC Nexera X2 system and Aeris Peptide XB-C18 column (50 mm 2.1 mm), × 3.6 µm bead diameter, at 27 ◦C. Eluent A: 0.1% formic acid in H2O, eluent B: 0.1% formic Int. J. Mol. Sci. 2020, 21, 3225 11 of 13 acid in MeCN. The gradient conditions (B%) were from 5% to 80% B within 40 min. Flow rate: 0.1 mL/min, injection volume: 5 µL. The MRM method was optimized automatically and the following transitions were chosen: 169LQTLEIPFHEIVTK182 [M + 2H]2+ (m/z 834.47) 834.47 242.10 → (b ), 834.47 970.60 (y ); for 213AVQFLVQTTMK223 [M + 2H]2+ (m/z 633.35) 633.35 72.10 (b ), 2 → 8 → 1 633.35 143.05 (a ); for 240SIAQDAK246 732.40 218.16 (y ), 732.40 461.26 (y ); for 292MIAAEAEK299 → 2 → 2 → 4 862.45 347.20 (y ), 862.45 547.28 (y ); for M(O)IAAEAEK 878.43 532.24 (b ), and 878.43 261.13 → 3 → 5 → 5 → (b ); for MIAAEAEK(TPP) [M + H]2+ 576.78 637.30 (y ) and 576.78 308.20. 2 → 3 → 5. Conclusions In conclusion, we demonstrated a new method of sensitive LC-MS-MRM analysis of podocin in urine sediments, using selective fixed charge tag derivatization of the ε amino group of C-terminal lysine residue in tryptic peptides. A peptide with the 292MIAAEAEK299 sequence, characteristic for both the canonical and short podocin isoform, was identified in urine samples from patients with PE, MN, and IgA nephropathy, both in native form and also as a TPP conjugate. The performed study revealed that the selected peptide is stable toward oxidation during the sample preparation steps. It may be assumed that the 292MIAAEAEK299 peptide sequence may serve as a marker of podocyturia, characteristic for different renal dysfunctions. The proposed strategy opens new possibilities for early diagnosis kidney injury. Cysteine-containing tryptic peptide enrichment, using commercially available TentaGel R RAM resin, was modified by the linker containing a maleimide reactive group toward thiol, according to the Michael addition mechanism. The captured peptides containing lysine residue at the C-terminus were modified on the ε-amino group by the quaternary ammonium group in the form of 2,4,6-triphenylpyrylium salt giving 2,4,6-triphenylpyridinium derivative. Such a methodology allowed the sensitive detection of cysteine-containing podocin tryptic peptide. The presented strategy may serve as a potential method in the investigations of PE biomarkers.

Author Contributions: The manuscript was written through contributions of all authors. R.B. performed all experiments and wrote the original draft; D.G. was responsible for peptide synthesis, urine sample preparation, method optimization; K.P.-L. and A.K. were responsible for clinical examination, urine sample collection, medical data analysis, and sample preparation. R.B., P.S., Z.S. and W.W. conceived and designed the project. All authors discussed the results and commented on the manuscript. All authors have read and agree to the published version of the manuscript. Funding: This research was supported by a grant No. UMO-2016/23/B/ST4/01036 from the National Science Centre, Poland. Acknowledgments: The authors would like to thank Wroclaw Center of Biotechnology, The Leading National Research Centre (KNOW) for years 2014–2018 and Andrzej Reszka (Shim-Pol, Poland) for providing access to the Shimadzu LCMS-8050 instrument. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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