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Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE

2015; Elsevier BV; Volume: 14; Issue: 8 Linguagem: Inglês

10.1074/mcp.m115.050518

ISSN

1535-9484

Autores

Arvind M. Korwar, Vannuruswamy Garikapati, Mashanipalya G. Jagadeeshaprasad, Ramesha H. Jayaramaiah, Shweta Bhat, Bhaskaran Regin, Sureshkumar Ramaswamy, Ashok P. Giri, Viswanathan Mohan, Muthuswamy Balasubramanyam, Mahesh J. Kulkarni,

Tópico(s)

Alcohol Consumption and Health Effects

Resumo

Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes. Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes. Diabetes is a complex metabolic disorder characterized by prolonged hyperglycemia resulting from defects in insulin secretion, insulin action, or both, leading to abnormalities in carbohydrate, fat, and protein metabolism (1.Assoc A.D. Standards of medical care in diabetes—2014.Diabetes Care. 2014; 37: S14-S80Crossref PubMed Scopus (3680) Google Scholar). According to the projection by the International Diabetes Foundation, around 592 million people will be affected by diabetes by the year 2040 (2.International Diabetes Federation.IDF Diabetes Atlas, 6th ed. Brussels, Belgium: International Diabetes Federation,. 2013; Google Scholar). Diabetes and its associated complications are becoming global public health problems and posing a serious challenge in disease management. Many studies have implicated advanced glycation end products (AGEs)1 in the development of insulin resistance, as well as in pathogenesis of diabetic complications (3.Manigrasso M.B. Juranek J. Ramasamy R. Schmidt A.M. Unlocking the biology of RAGE in diabetic microvascular complications.Trends Endocrin. Met. 2014; 25: 15-22Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The levels of AGEs increase substantially in diabetic plasma due to the hyperglycemic condition. Factors such as oxidative stress, overnutrition, and foods rich in glycating agents promote the formation of AGEs even in nondiabetic condition (4.Monnier V.M. Toward a Maillard reaction theory of aging.Prog. Clin. Biol. Res. 1989; 304: 1-22PubMed Google Scholar). Oral AGEs foster insulin resistance and diabetes by down-regulation of anti-AGE receptor-1(AGER1), sirtuin 1, and up-regulation of receptor for AGEs (RAGE) (5.Uribarri J. Cai W. Ramdas M. Goodman S. Pyzik R. Chen X. Zhu L. Striker G.E. Vlassara H. Restriction of advanced glycation end products improves insulin resistance in human type 2 diabetes potential role of ager1 and sirt1.Diabetes Care. 2011; 34: 1610-1616Crossref PubMed Scopus (247) Google Scholar). AGEs affect glucose uptake, transport and promote insulin resistance in adipocytes (6.Wu C.H. Huang H.W. Huang S.M. Lin J.A. Yeh C.T. Yen G.C. AGE-induced interference of glucose uptake and transport as a possible cause of insulin resistance in adipocytes.J. Agr. Food Chem. 2011; 59: 7978-7984Crossref PubMed Scopus (23) Google Scholar). While in skeletal muscle cells AGEs inhibit insulin action, mediated through RAGE (7.Cassese A. Esposito I. Fiory F. Barbagallo A.P. Paturzo F. Mirra P. Ulianich L. Giacco F. Iadicicco C. Lombardi A. Oriente F. Van Obberghen E. Beguinot F. Formisano P. Miele C. In skeletal muscle advanced glycation end products (AGEs) inhibit insulin action and induce the formation of multimolecular complexes including the receptor for AGEs.J. Biol. Chem. 2008; 283: 36088-36099Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The AGE-RAGE axis induces oxidative stress, activates proinflammatory pathways and has been considered as a principal pathway in the pathogenesis of diabetes and its complications (8.Yan S.D. Schmidt A.M. Anderson G.M. Zhang J. Brett J. Zou Y.S. Pinsky D. Stern D. Enhanced cellular oxidant stress by the interaction of advanced glycation end-products with their receptors binding-proteins.J. Biol. Chem. 1994; 269: 9889-9897Abstract Full Text PDF PubMed Google Scholar). AGE interacts with RAGE in different cells and tissues, contributing to pathogenesis in diabetes (9.Kulkarni M.J. Korwar A.M. Mary S. Bhonsle H.S. Giri A.P. Glycated proteome: From reaction to intervention.Proteom. Clin. Appl. 2013; 7: 155-170Crossref PubMed Scopus (26) Google Scholar). By and large, AGEs contribute to development of insulin resistance leading to diabetes, as well as in the pathogenesis of diabetic complications. Therefore, analysis of plasma AGEs can possibly provide information about the severity of diabetes. Human serum albumin (HSA), one of the most abundant plasma proteins, is highly glycated and contributes predominantly to the plasma AGEs. Apart from its role in pathogenesis, AGE-modified HSA (AGE-HSA) has been suggested as an alternative diagnostic marker to glycated hemoglobin (HbA1c) for monitoring glycemic status in diabetes (10.Juraschek S.P. Steffes M.W. Selvin E. Associations of alternative markers of glycemia with hemoglobin A(1c) and fasting glucose.Clin. Chem. 2012; 58: 1648-1655Crossref PubMed Scopus (69) Google Scholar). Although HbA1c is considered the "gold standard" marker, reflecting the glycemic status over the period of 8–10 weeks (1.Assoc A.D. Standards of medical care in diabetes—2014.Diabetes Care. 2014; 37: S14-S80Crossref PubMed Scopus (3680) Google Scholar, 10.Juraschek S.P. Steffes M.W. Selvin E. Associations of alternative markers of glycemia with hemoglobin A(1c) and fasting glucose.Clin. Chem. 2012; 58: 1648-1655Crossref PubMed Scopus (69) Google Scholar), factors like anemia, blood loss, splenomegaly, and iron deficiency affect HbA1c levels (11.Radin M.S. Pitfalls in hemoglobin A1c measurement: When results may be misleading.J. Gen. Intern. Med. 2014; 29: 388-394Crossref PubMed Scopus (161) Google Scholar). AGE-HSA reflects glycemic status over the preceding 3–4 weeks and has been recommended in gestational diabetes (12.Hashimoto K. Osugi T. Noguchi S. Morimoto Y. Wasada K. Imai S. Waguri M. Toyoda R. Fujita T. Kasayama S. Koga M. A1C but not serum glycated albumin is elevated because of iron deficiency in late pregnancy in diabetic women.Diabetes Care. 2010; 33: 509-511Crossref PubMed Scopus (84) Google Scholar). In diabetes, the levels of AGE-HSA increase and were found to be positively correlated with hyperglycemia (13.Juraschek S.P. Steffes M.W. Miller 3rd, E.R. Selvin E. Alternative markers of hyperglycemia and risk of diabetes.Diabetes Care. 2012; 35: 2265-2270Crossref PubMed Scopus (66) Google Scholar, 14.Bhonsle H.S. Korwar A.M. Kote S.S. Golegaonkar S.B. Chougale A.D. Shaik M.L. Dhande N.L. Giri A.P. Shelgikar K.M. Boppana R. Kulkarni M.J. Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes.J. Proteome Res. 2012; 11: 1391-1396Crossref PubMed Scopus (66) Google Scholar). In addition, several recent studies have suggested that the levels of AGE-HSA are associated with prediabetic condition (15.Chan C.L. Pyle L. Kelsey M. Newnes L. Zeitler P.S. Nadeau K.J. Screening for type 2 diabetes and prediabetes in obese youth: Evaluating alternate markers of glycemia—1,5 anhydroglucitol, fructosamine, and glycated albumin.Pediatr. Diabetes. Epub ahead of print. 2015; Google Scholar) and microalbuminuria (16.Donnelly S.M. Accumulation of glycated albumin in end-stage renal failure: Evidence for the principle of "physiological microalbuminuria.".Am. J. Kidney Dis. 1996; 28: 62-66Abstract Full Text PDF PubMed Scopus (14) Google Scholar). Therefore, quantification of AGE-HSA is of utmost clinical significance. Thus, understanding the site-specific modification and their dynamic transformation to heterogeneous AGEs is quite critical for mass spectrometric quantification. AGEs can be quantified by various approaches, including colorimetric assay, ketoamine oxidase assay, enzyme-linked boronate immunoassay, fluorescence spectroscopy, boronic acid affinity chromatography assay, and mass spectrometry (MS) (17.Capote F. Sanchez J.C. Strategies for proteomic analysis of non-enzymatically glycated proteins.Mass Spectrom. Rev. 2009; 28: 135-146Crossref PubMed Scopus (39) Google Scholar). Among these approaches, MS offers precise characterization of protein glycation, including the amino acid involved in the modification. Most of the AGEs reported in vitro and in vivo were discovered by MS-based techniques (18.Arena S. Salzano A.M. Renzone G. D'Ambrosio C. Scaloni A. Non-enzymatic glycation and glycoxidation protein products in foods and diseases: An interconnected, complex scenario fully open to innovative proteomic studies.Mass Spectrom. Rev. 2014; 33: 49-77Crossref PubMed Scopus (63) Google Scholar). AML modification has been extensively studied by different MS approaches. The fragmentation pattern and diagnostic ions for AML rearrangement product has been well established (19.Yaylayan V. Sporns P. Diagnostic ion series for the identification of Amadori rearrangement products by MS techniques based on electron-impact ionization.J. Agr. Food Chem. 1989; 37: 978-981Crossref Scopus (13) Google Scholar, 20.Lapolla A. Fedele D. Reitano R. Aricò N.C. Seraglia R. Traldi P. Marotta E. Tonani R. Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides.J. Am. Soc. Mass Spectrom. 2004; 15: 496-509Crossref PubMed Scopus (155) Google Scholar). Further specific neutral loss ions of 162 Da, 120 Da, and 84 Da and water loss of 36 Da arising from hexose moiety of glycated peptide were also considered as signature ions to validate the glycation of peptides in HSA (21.Brancia F.L. Bereszczak J.Z. Lapolla A. Fedele D. Baccarin L. Seraglia R. Traldi P. Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer.J. Mass Spectrom. 2006; 41: 1179-1185Crossref PubMed Scopus (31) Google Scholar, 22.Gadgil H.S. Bondarenko P.V. Treuheit M.J. Ren D. Screening and Sequencing of glycated proteins by neutral loss scan LC/MS/MS method.Anal. Chem. 2007; 79: 5991-5999Crossref PubMed Scopus (64) Google Scholar). Similar characteristic patterns of water loss (18, 36, and 54 Da) ions and immonium ions derived from lysine arising from AML-modified peptide were also used to identify glycated peptides (23.Frolov A. Hoffmann P. Hoffmann R. Fragmentation behavior of glycated peptides derived from D-glucose, D-fructose and D-ribose in tandem mass spectrometry.J. Mass Spectrom. 2006; 41: 1459-1469Crossref PubMed Scopus (94) Google Scholar, 24.Stefanowicz P. Kapczynska K. Jaremko M. Jaremko Ł. Szewczuk Z. A mechanistic study on the fragmentation of peptide-derived Amadori products.J. Mass Spectrom. 2009; 44: 1500-1508Crossref PubMed Scopus (13) Google Scholar). Diagnostic ions serve as the most reliable way of identifying glycated peptide by tandem mass spectrometry. Thus, having a good MS/MS fragment ion is key for precise characterization of glycation. However, the ratio of in vivo AGE-modified to unmodified protein is significantly low, which limits better MS/MS. Therefore, to achieve efficient identification, enrichment of glycated peptides using boronate affinity chromatography (BAC) was adopted prior to MS analysis (25.Frolov A. Hoffmann R. Analysis of Amadori peptides enriched by boronic acid affinity chromatography.Ann, NY Acad. Sci. 2008; 1126: 253-256Crossref PubMed Scopus (43) Google Scholar). Further, by using a combination of immunodepletion, enrichment and fractionation strategies, a total of 7,749 unique glycated peptides corresponding to 1,095 native human plasma proteins, 1,592 in vitro glycated human plasma proteins, and 1,664 erythrocyte proteins were identified (26.Zhang Q. Monroe M.E. Schepmoes A.A. Clauss T.R. Gritsenko M.A. Meng D. Petyuk V.A. Smith R.D. Metz T.O. Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects.J. Proteome Res. 2011; 10: 3076-3088Crossref PubMed Scopus (83) Google Scholar). In these lines, we have previously reported a database search approach for the identification of glycated peptide in a crude or nonenriched sample by untargeted MS/MS or data-independent workflow (27.Bhonsle H.S. Korwar A.M. Kesavan S.K. Bhosale S.D. Bansode S.B. Kulkarni M.J. "Zoom-In"—A targeted database search for identification of glycation modifications analyzed by untargeted tandem mass spectrometry.Eur. J. Mass Spectrom. 2012; 18: 475-481Crossref PubMed Scopus (14) Google Scholar). Glycation is chronic process; a given protein can undergo dynamic heterogeneous transformations as these proteins have varying biological lifespans, influencing the function of a protein. Thus, to assess the degree of glycation at a given pathophysiological condition, precise identification of glycation becomes critical. In this regard, a stable-isotope-dilution tandem mass spectrometry method was employed for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins (28.Teerlink T. Barto R. Ten Brink H.J. Schalkwijk C.G. Measurement of N-epsilon-(carboxymethyl)lysine and N-epsilon-(carboxyethyl)lysine in human plasma protein by stable-isotope-dilution tandem mass spectrometry.Clin. Chem. 2004; 50: 1222-1228Crossref PubMed Scopus (114) Google Scholar), and 13C6-glucose was utilized to quantify glycated proteins in the plasma and erythrocytes (29.Priego-Capote F. Ramírez-Boo M. Hochstrasser D. Sanchez J.C. Qualitative and quantitative analysis of glycated proteins in human plasma by glucose isotopic labeling with 13C6-reducing sugars.Methods Mol. Biol. 2011; 728: 219-232Crossref PubMed Scopus (3) Google Scholar, 30.Priego-Capote F. Ramirez-Boo M. Hoogland C. Scherl A. Mueller M. Lisacek F. Sanchez J.C. Human hemolysate glycated proteome.Anal. Chem. 2011; 83: 5673-5680Crossref PubMed Scopus (17) Google Scholar). In a recent study, the glycation-sensitive peptides of HSA that could serve as markers for early diagnosis of type 2 diabetes were quantified by using an MS-based 18O-labeling technique (31.Zhang M. Xu W. Deng Y.L. A New Strategy for Early Diagnosis of Type 2 Diabetes by standard-free, label-free LC-MS/MS quantification of glycated peptides.Diabetes. 2013; 62: 3936-3942Crossref PubMed Scopus (30) Google Scholar). However, most of the previous studies have focused on AML modification, rather than other AGE modification. In fact, CML and CEL are the predominant AGEs, constituting up to 80% of total AGEs (32.Reddy S. Bichler J. Wells-Knecht K.J. Thorpe S.R. Baynes J.W. N-epsilon-(carboxymethyl)lysine is a dominant advanced glycation end-product (AGE) antigen in tissue proteins.Biochemistry. 1995; 34: 10872-10878Crossref PubMed Scopus (465) Google Scholar, 33.Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (0) Google Scholar). Diagnostic reporter ions for CML and CEL were reported recently by Prof. Ralf Hoffmann's group (34.Greifenhagen U. Nguyen V.D. Moschner J. Giannis A. Frolov A. Hoffmann R. Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma.J. Proteome Res. 2015; 14: 768-777Crossref PubMed Scopus (30) Google Scholar). Here, for the first time, we report comprehensive development of an MS/MS fragment ion library for AML, CML, and CEL modifications of albumin. Further, fragment ion library was used as reference for quantification of AML-, CML-, and CEL-modified peptides of albumin in clinical plasma of healthy, prediabetic, diabetic, and microalbuminuria. Targeted SWATH analysis has led to quantification of 13 glycated peptides representing nine lysine sites. These peptides could serve as novel markers in diabetes. Subjects were recruited from Dr. Mohan's Diabetes Specialties Centre, Chennai, India. The study was approved by the institutional ethics committee of Madras Diabetes Research Foundation, and prior written informed consent was obtained from all the study subjects. The study was performed in accordance to the Helsinki Declaration. Healthy control, prediabetic, diabetic, and microalbuminuria patients other than known history of cancer, hematuria, hypothyroidism, and history of any known infection or inflammatory diseases were included in this study. Peripheral blood was collected in EDTA vacutainers (BD Biosciences, USA). The plasma was separated by centrifugation at 1500 × g for 15 min and aliquots were stored at −80 °C. The biochemical analyses were performed immediately after the sample collection. Descriptive characters and diagnostic parameters, including fasting blood glucose, HbA1c, oral glucose tolerance test, postprandial blood sugar, lipids, urea, creatinine, and microalbumin were measured (Supplemental Table 1). The fasting plasma samples were categorized into four groups, viz., from healthy control subjects (normal glucose tolerance (NGT) ], prediabetics [ impaired glucose tolerance (IGT) ], patients with type 2 diabetes mellitus [ T2DM ], and diabetics with microalbuminuria [MIC) as per the World Health Organization Consulting Group Criteria. Equal volumes of two plasma samples with similar HbA1c (deviation of < 0.2%) were pooled, and three such pooled plasma in each group were used for mass spectrometric technical triplicate analysis. Overview of complete study design is shown in Fig. 1. First, the AGE-HSA MS/MS fragment ion library with diagnostic ions was established for glycation modifications AML, CML, and CEL. Diagnostic signature ions were validated for glycated peptides in diabetic plasma using targeted parallel reaction monitoring workflow. Further, based on the fragment ion library information, glycated peptides in healthy control, prediabetic, diabetic, and microalbuminuria plasma were quantified by using SWATH to discover candidate peptide biomarkers to assess the extent of glycation in diabetes. The results of SWATH were verified by MSE, another label-free quantification method. Two-way analysis of variance (ANOVA) was performed to assess the statistical significance of quantified glycated peptides. All the chemicals were procured from Sigma-Aldrich (Sigma-Aldrich, MO, USA). MS-grade solvents (acetonitrile (ACN), water, and methanol) were procured from J T. Baker (J T. Baker, PA, USA). RapiGest was procured from Waters (Waters Corporation, MA, USA). Membrane filters of 3 and 30 KDa cut off were procured from Millipore (Millipore, MA, USA). AGE-HSA was synthesized as reported elsewhere (35.Bhonsle H.S. Singh S.K. Srivastava G. Boppana R. Kulkarni M.J. Albumin competitively inhibits glycation of less abundant proteins.Protein Peptide Letters. 2008; 15: 663-667Crossref PubMed Scopus (22) Google Scholar, 36.Ikeda K. Higashi T. Sano H. Jinnouchi Y. Yoshida M. Araki T. Ueda S. Horiuchi S. N-epsilon-(carboxymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction.Biochemistry. 1996; 35: 8075-8083Crossref PubMed Scopus (412) Google Scholar). Briefly, HSA (50 mg/ml) was dissolved in 10 ml of 0.2 m sodium phosphate buffer (pH 7.4) containing 0.5 m of glucose and 0.05% sodium azide, sterilized by ultrafiltration (0.22 μm filter), and incubated for 7 days at 37 °C. The samples were then extensively washed with PBS using 30 kDa cut-off filters. CML- and CEL-modified HSA was synthesized as reported elsewhere (36.Ikeda K. Higashi T. Sano H. Jinnouchi Y. Yoshida M. Araki T. Ueda S. Horiuchi S. N-epsilon-(carboxymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction.Biochemistry. 1996; 35: 8075-8083Crossref PubMed Scopus (412) Google Scholar, 37.Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. Cloning and expression of a cell-surface receptor for advanced glycosylation end-products of proteins.J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar). Briefly, HSA (50 mg/ml) and sodium cyanoborohydride (0.15 m) were dissolved in 0.2 m sodium phosphate buffer (pH 7.4), to which either glyoxylic acid (GA) (0.045 m) or methylglyoxal (MG) (0.05 m) was added to a final volume of 10 ml for the synthesis of CML- or CEL-modified HSA, respectively. The solution was incubated for 24 h at 37 °C, followed by washing with PBS using 30 kDa cut-off filters. In-solution tryptic digestion: Equal amounts of protein (100 μg) of AGE-modified HSA and clinical plasma proteins were diluted with 100 μl of ammonium bicarbonate buffer (50 mm) containing 0.1% RapiGest, followed by incubation at 80 °C for complete proteome solubilization. The denatured proteins were then reduced with DTT (0.1 m) at 60 °C for 15 min, followed by alkylation with iodoacetamide (0.2 m) at room temperature in the dark for 30 min. The proteins were digested with proteomic grade trypsin at 1:50 enzyme to substrate ratio overnight at 37 °C. The digestion reaction was stopped by adding concentrated HCl and incubated for 10 min at 37 °C before being centrifuged. The peptides were desalted by using C18 Zip tip (Millipore, MA, USA) and concentrated by vacuum centrifuge and stored at −20 °C until further use. Instrument-specific methods and settings [(LC-HR/AM Q-Exactive Orbitrap and parallel reaction monitoring), (Triple TOF 5600 (DDA and SWATH-MS), (label-free LC-MSE on SYNAPT HDMS)] used for the construction of the fragment ion library and quantification of glycated peptides are described below. Peptide digests (1.5 μg) were separated by using Accela 1250 UHPLC (Thermo Fisher Scientific) equipped with a Hypersil Gold C18-reverse phase column (150*2.1 mm, 1.9 μm). The sample was loaded onto the column with 98% of mobile phase A (100% water, 0.1% formic acid (FA)) and 2% of mobile phase B (100% ACN, 0.1% FA) at 350 μl/min flow rate. Peptides were eluted with a 45 min linear gradient of 2 to 40% mobile phase B. In case of plasma samples, the LC method was extended to 120 min with a linear gradient of 2 to 50% of mobile phase B. The column temperature was set to 40 °C and auto sampler at 4 °C. All samples were analyzed on hybrid quadruple Q-Exactive Orbitrap MS. The instrument tune parameters were optimized for the better results as: spray voltage 4,200 V, capillary temperature 320 °C, heater temperature 200 °C, S-lens RF value 55, sheath and auxiliary gases pressure were 30 and 8 psi, respectively. The samples were acquired in positive ionization mode in data-dependent manner using a top-five method with scan range from 350–1,800 m/z. MS spectra were acquired at a resolution of 70,000 with maximum injection time (IT) of 120 ms and automatic gain control (AGC) value of 1 × e6 ions; MS/MS spectra were acquired at 17,500 resolution with maximum IT of 120 ms and AGC value of 1 × e5 ions. Precursor's selectivity was performed at an isolation width of 3 m/z, under fill ratio of 0.3%, and dynamic exclusion time of 15 s. The peptide fragmentation was performed in high energy collision induced dissociation (HCD) cell using normalized HCD at 30 eV. Peptide digests were acquired with optimized tune parameters in targeted/dd-MS2 mode by using glycated peptide ion m/z inclusion list (Supplemental Table 2). The isolation width was set to 1 m/z for the selection of precursor. MS spectra were acquired at a resolution of 70,000 with maximum IT of 120 ms and AGC value of 5 × e4 ions. The peptide fragmentation was performed at a resolution of 17,500, maximum IT of 120 ms, AGC value of 2 × e4 ions, and normalized HCD at 30 eV. LC-HR/AM Q-Exactive Orbitrap mass spectrometric data were processed by using Proteome Discoverer, Version 1.4.0.288, (Thermo Fisher Scientific). The SEQUEST HT (a computer algorithm for database search) was used for peptide identification. The data were searched against HSA protein database (P02768-UniProt). The search was performed using the following parameters: Peptide and fragment mass tolerance were 10 ppm, 0.5 Da, respectively, with two missed cleavages and 1% false discovery rate. Search criteria included fixed and variable modifications as carbamidomethylation (C) and oxidation (M), respectively. Additional variable lysine-specific glycation modifications AML (+162.02); CML (+58.005 Da), and CEL (+72.021 Da) were considered. Peptide digests (3 μg) were separated by using an Eksigent MicroLC 200 system (Eksigent, Dublin, CA) equipped with Eksigent C18-reverse phase column (100*0.3 mm, 3 μm, 120 Å). The sample was loaded onto the column with 97% of mobile phase A (100% water, 0.1% FA) and 3% of mobile phase B (100% ACN, 0.1% FA) at 8 μl/min flow rate. Peptides were eluted with a 120 min linear gradient of 3 to 50% mobile phase B. The column temperature was set to 40 °C and auto sampler at 4 °C. The same chromatographic conditions were used for both DDA and SWATH acquisition. All samples were analyzed on AB-SCIEX 5600 Triple TOF mass spectrometer in positive and high-sensitivity mode. The dual source parameters were optimized for better results: ion source gases GS1, GS2, curtain gas at 25 psi, temperature 200 °C, and ion spray voltage floating at 5,500 V. The accumulation time in full scan was 250 ms for a mass range of 350–1,800 m/z. The parent ions are selected based on the following criteria: ions in the MS scan with intensity more than 120 counts per second, charge stage between +2 to +5, and mass tolerance 50 mDa. Ions were fragmented in the collision cell using rolling collision energy (CE) with an additional CE spread of ± 15 eV. Glycated peptides derived from synthetically glycated HSA were acquired in technical triplicate using the above-mentioned DDA method. DDA mass spectrometric files were searched using ProteinPilot software, Version 4.0.8085 (AB SCIEX, MA, USA) with the Paragon algorithm against human serum albumin protein database (P02768-UniProt) at 1% false discovery rate. The ProteinPilot output file (.group) was used as a standard peptide spectral library. In SWATH-MS mode, the instrument was specifically tuned to optimize the quadrupole settings for the selection of precursor ion window of 25 m/z wide. Using an isolation width of 26 m/z (containing 1 m/z for the window overlap), a set of 34 overlapping windows was constructed covering the precursor mass range of 400–1,250 m/z. SWATH MS/MS spectra were collected from 100 to 2,000 m/z. Ions were fragmented in the collision cell using rolling collision energy with an additional CE spread of ± 15 eV. An accumulation time (dwell time) of 96 ms was used for all fragment-ion scans in high-sensitivity mode, and for each SWATH-MS cycle a survey scan in high-resolution mode was acquired for 100 ms, resulting in a duty cycle of 3.33 s. The source parameters were similar to that of DDA acquisition. SWATH analysis was performed for three biological replicates and technical triplicates each from healthy control, prediabetic diabetic, and microalbuminuria. Each biological replicate was a pool of two plasma samples with similar HbA1c. The spectral alignment and targeted data extraction of SWATH-MS data were performed using Peakview software, Version 1.2.03 (AB SCIEX, MA, USA). The peptide data (.MRKVW) files were used for quantification of glycated peptides of HSA using Markerview software, Version 1.2.1.1 (AB SCIEX, MA, USA). Normalization was performed using total area sum. The peptides with a p value ≤ 0.05 were considered for quantification. Protein digest (400 ng) was analyzed by LC-MSE workflow using nano

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