Carboxyl-Ester Lipase Maturity-Onset Diabetes of the Young Disease

Article pubs.acs.org/jpr

Carboxyl-Ester Lipase Maturity-Onset Diabetes of the Young Disease Protein Biomarkers in Secretin-Stimulated Duodenal Juice Yngvild Bjorlykke,†,‡ Heidrun Vethe,†,‡,∥ Marc Vaudel,§,∥ Harald Barsnes,§ Frode S. Berven,§ Erling Tjora,‡ and Helge Raeder*,†,‡ †

KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Jonas Lies Vei 65, Bergen 5021, Norway ‡ Department of Pediatrics, Haukeland University Hospital, Jonas Lies vei 65, Bergen 5021, Norway § Proteomics Unit (PROBE), Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Bergen 5009, Norway S Supporting Information *

ABSTRACT: Patients with carboxyl-ester lipase-maturity-onset diabetes of the young (CEL-MODY) display distinct disease stages toward the development of monogenic diabetes and exocrine pancreatic disease. The finding of differentially increased proteins, some related to MAPK signaling, in a discovery proteomics study of secretin-stimulated duodenal juice in three CELMODY patients, prompted us to monitor their abundance in an extensive number of CEL-MODY subjects at different disease stages and controls using targeted proteomics. In the current study, we demonstrate the feasibility of selected reaction monitoring assays to quantify protein levels in secretinstimulated duodenal juice. Furthermore, we define a set of five peptides for potential use as diagnostic tests in CEL-MODY patients. Finally, we propose a further set of seven proteins with a likely pathogenic role in CEL-MODY disease progression.

KEYWORDS: CEL-MODY, diabetes, biomarker, proteomics, duodenal juice, mass spectrometry, selected reaction monitoring

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INTRODUCTION

mics with an additional isolation and fragmentation event (MS3 scan)7 in three diabetic family members with CEL-MODY. There, we demonstrated that their pancreatic secretome contained a distinct protein signature of increased proteins, including mitogen-activated protein kinase (MAPK) target proteins. In the present study, we have developed selected reaction monitoring (SRM) assays based on the most differentially increased proteins detected among the CELMODY patients,3 and compared their performance as biomarkers in secretin-stimulated duodenal juice from 8 nondiabetic, 8 diabetic CEL-MODY patients, and 21 unrelated controls. SRM is the method of choice8 for targeted proteomic quantification.9−12 SRM has been used successfully in several biological fluids such as plasma,13 cerebrospinal fluid (CSF),14 and urine15,16 but to our knowledge never in secretinstimulated duodenal juice,17 which contains high amounts of proteases and is a promising proximal fluid4 for the studies of

Carboxyl-ester lipase maturity-onset diabetes of the young (CEL-MODY) is an excellent human monogenic disease model for pancreatic disease since patients display distinct disease stages starting in early childhood with pancreatic exocrine dysfunction (measured by low fecal elastase levels1) and pancreatic lipomatosis2 and followed in their fourth decade by diabetes development due to beta cell dysfunction1 and signs of pancreatic malabsorption1 and multiple pancreatic cyst formation.3 Clearly, diabetes development heralds a more severe and advanced disease stage in these patients. Because CEL mutation carriers develop age-dependent disease stages in such a predictable manner, studies of protein biomarker levels in these CEL mutation carriers before and after diabetes development may provide important information about pathogenic proteins as well as provide potential diagnostic and therapeutic proteins markers in general4 to be further explored more generally in pancreatic disease. Pancreatic diseases include severely debilitating diseases where preclinical biomarkers may have profound clinical impact such as diabetes5 and pancreatic cancer.6 In a recent discovery study,3 we investigated secretinstimulated duodenal juice with multiplexed MS-based proteo© 2014 American Chemical Society

Special Issue: Environmental Impact on Health Received: July 18, 2014 Published: October 21, 2014 521

dx.doi.org/10.1021/pr500750z | J. Proteome Res. 2015, 14, 521−530

Journal of Proteome Research

Article

pancreatic diseases.18 Here we demonstrate the applicability of SRM to secretin-stimulated duodenal juice and confirm and extend our findings from the discovery proteomics study, hence providing potential diagnostic and pathogenic markers for the distinct disease stages of pancreatic disease development in CEL-MODY patients.

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amount in the sample. The samples were incubated for 16 h at 37 °C. As a final step, 15 μL of 10% FA was added to quench the reaction. Design of SRM Assays

SRM assays were designed for 22 proteins (21 biomarker candidates and amylase) using synthetic peptides.21 Signature peptides (at least three per protein) were defined and ordered in crude quality (as synthesized by the manufacturer and with an average purity of 50% according to the manufacturer) from Thermo Scientific based on observed peptides in the discovery TMT study3 and Peptide Atlas (http://www.peptideatlas.org). Skyline22 was used to define optimized transitions (at least five transition) per peptide in addition to building the method. Peptides were optimized on a Q-Trap 5500 instrument (AB SCIEX, Framingham, MA) coupled to a Dionex Ultimate 3000RS nano-LC with a precolumn (Dionex, Acclaim PepMap Nano Trap column, C18, 75 μm i.d. × 2 cm, 3 μm) and a custom-made analytical column (Dionex, Acclain PepMap100 RSLCnano column, 75 μm i.d. × 15 cm, C18, 2 μm). The LC was operated with a flow rate of 250 nL/min over 70 min with a solvent A consisting of 0.1% FA and a solvent B consisting of 0.1% FA and 90% ACN. Peptides were eluted with a gradient of 5−90% solvent B over 45.5 min, held at 90% solution B for 6 min, then ramped down to 5% solvent B in 3.5 min, and finally held at 5% solvent B the last 15 min. The MS run time was 68 min. Both normal SRM mode and scheduled SRM mode were used during optimization. Optimally, the SISs (Stable Isotope Standards) peptides should be added to the sample in amounts corresponding to the endogenous peptide levels. For this purpose, the SISs were further optimized using different concentrations of SISs analyzed in a matrix of duodenal juice from a pool of 10 CEL mutation carriers. A standard curve was drawn to find the concentration where the SIS peptide was in a 1:1 relative level to the target endogenous peptide.

MATERIALS AND METHODS

Chemicals

Secretin (Secrelux) was purchased from Sanochemia Diagnostics (Neuss, Germany) and protease inhibitor (cOmplete) from Roche Diagnostics (Mannheim, Germany). Urea, D,Ldithiothreitol for electrophoresis (DTT), iodoacetamide (IAA), and calcium chloride (CaCl2) were purchased from SigmaAldrich (St. Louis, MO). Tris(hydromimethyl) amonimethane and bicinchoninic acid assay (BCA) protein assay measurement kits were purchased from Merck KGaA (Darmstadt, Germany). Trypsin porcine was purchased from Promega (Madison, WI). Water, acetonitrile (ACN), and formic acid, all MS-grade, were purchased from Sigma-Aldrich. Stable isotope standards (SISs) were purchased from Thermo Fisher Scientific (Ulm, Germany). Biological Material

Secretin-stimulated duodenal juice from CEL mutation carriers and controls were obtained by regular gastroscopy at the Department of Medicine, Haukeland University Hospital, Bergen, Norway. Secretin (Secrelux) was administered intravenously 30 min before gastroscopy (1 clinical unit (CU)/kg, maximum dose 70 CU). The endoscope was placed distal to papilla vateri, and duodenal juice was collected through the suctioning channel of the endoscope. Juice was sampled in three portions of 5 min collection time. Protease inhibitor (0.2 mL solution of one tablet per 1.5 mL of water added per mL of duodenal juice) was added before the samples were snap-frozen on liquid nitrogen. Diabetes was defined according to the WHO criteria.19 All studies were approved by The Regional Committee for Medical and Health Research Ethics (REK#2010/198) and performed according to the Helsinki Declaration. Written informed consent was obtained from all subjects or their parents. The patients were of Northern European descent and were recruited from the Norwegian MODY Registry.

Sample Cleanup and SIS Spike-in

Sample cleanup was performed using a reverse-phase Oasis HLB μElution Plate 30 μm (Waters, Milford, MA). In brief, the plate was washed once with 500 μL of 80% ACN/0.1% FA and subsequently washed twice with 500 μL of 0.1% FA. The samples together with synthetic heavy peptide mixture were added to the wells on the μElution plate, followed by washing three times with 500 μL of 0.1% FA. To elute the bound peptides, 100 μL of 80% ACN/0.1% FA was added twice. The centrifuge speed was 200g for 1 min for all steps, except the step concerning sample addition, where 150g was used for 3 min. Centrifugation between all steps was performed with a Biofuge Statos centrifuge (Heraeus, Buckinghamshire, U.K.).

Immunometric Methods

Alpha-amylase activity was determined with the colorimetric end-point assay Phadebas Amylase test (Magle AB, Lund, Sweden) as previously described.20 Sample Preparation and Protein Digestion

Total protein concentration in samples of secretin-stimulated duodenal juice was measured using a BCA protein assay measurement kit. Duodenal juice corresponding to 10 μg was denatured in 20 μL of urea (8 M) followed by adding 20 μL of trypsin buffer (100 mM EDTA/50 mM Tris/1 mM CaCl2) and a 5 min incubation in an Eppendorf mixer. Reduction (100 mM DTT) and alkylation (200 mM IAA) were performed, both steps followed by a 1 h incubation in, respectively, room temperature and room temperature under dark conditions. To avoid unwanted alkylation, we added 0.8 μL of 100 mM DTT, followed by 10 min of incubation in an Eppendorf mixer. Urea concentration was adjusted to 1 M by adding 110.2 μL of trypsin buffer. Trypsin was prepared in a stock solution (0,5 μg/μL) and added at a ratio of 1:50 (w/w) to the protein

Sample Fractionation

All samples were fractionated using mixed-mode (MM) reverse-phase weak anion exchange (RP-WAX) connected to an Agilent Technology 1260 off-line LC system. A Promix mixed-phase (MP) 250 mm × 2.1 mm id, pore size 300 Å column (SIELC Technologies, Prospect Heights, IL) was used. In brief, the desalted and dried samples were resuspended in 120 μL of buffer A (20 mM Ammonium formate/3% ACN, pH 6.5) and loaded onto the column. Column flow was set to 50 μL/min and the gradient length was 70 min. From 0 to 45 min, buffer B (2 mM Ammonium formate/80% ACN) increased linearly from 15 to 60%, from 45 to 55 min 60% B, from 55 to 65 min 100% B, and from 65 to 70 min 15% B. Sample 522



Article

Figure 1. (A) Workflow diagram. The current study was a targeted proteomics approach to secretin-stimulated duodenal juice based on findings from a previous quantitative proteomics discovery study in CEL mutation carriers.3 The figure is adapted from ref 1 and shows the clinically characterized part of the family with monogenic CEL-MODY disease from which we sampled secretin-stimulated duodenal juice from eight diabetic and eight prediabetic CEL mutation carriers for the targeted proteomics studies. The selected targeted proteomics peptides were based on the most differentially expressed proteins from the discovery study.3 The healthy controls were unrelated to the family. (B) Amylase quantities are measured with SRM by the signature peptide TGSGDIENYNDATQVR for healthy controls and CEL mutation carriers. The SRM quantities are measured as femtamol/μL, and the calculations are explained in the Methods section. P value 0.0041, whiskers showing median with standard deviation. (C) Correlation plot of SRM measurements and enzyme activity measurements for amylase in 19 healthy controls and 13 CEL mutation carriers. The R2 value is 0.55.

SRM Data Analysis

collection was performed from 0 to 70 min, where a total of seven fractions were collected with 10 min intervals. Fractions 1−5 were frozen, freeze-dried, and dissolved (3%ACN/5%FA) to be further processed for SRM analysis.

The SRM data were analyzed by manual inspection in Skyline,22 and the area under curve for the most abundant transition per peptide was used for quantification. Given that we loaded onto the precolumn 1 μg total protein amount of each sample and each standard spike-in (SIS) was approximately 1:1 to the endogenous peptide, the signal readout of the ratio of sample (light peptide) to SIS (heavy peptide) was multiplied by the concentration of the SIS (0.5 fmol/μg) and further multiplied by the initial total protein concentration (μg/ μL) to yield the quantity of the sample as fmol/μL. We used fmol/μL to be able to correlate SRM quantities with the enzymatic activities (measured as units/μL).

Stable Isotope Dilution SRM MS Analysis

For LC−MS analysis, a Q-Trap 5500 coupled to a Dionex Ultimate 3000RS nano-LC system was used. The concentrated samples were resuspended in 8 μL (3% ACN/5% FA), where 6 uL (1 μg) was loaded onto the precolumn. Instrument setup, gradient, and solutions were the same as when designing the assays. The curtain gas was set to 30. The Gas1/nebulizer was set to 12. The interface temperature was 150 °C. The target scan time was 1 s, and the detection window was 240 s for the scheduled SRM. Three to five transitions per peptide were monitored, where the most intense transition was selected for relative quantification.

Statistics

The peptide relative concentration between patient and control was calculated using the median protein abundance for each group. The Mann−Whitney U test was used to find significant differences between groups. Pearson correlation and Mann−

SRM Data Availability

The SRM data were deposited to the Panorama repository.23 523



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Table 1. Demographic Tablea discovery (previous) study diabetic mutation carrier number of patients female/total (n) age at juice collection (years) age at diabetes diagnosis (years) HbA1C (%) amylase activity (U/mL) bicarbonate (mEq/L)

validation (current) study

CONTROL

3 0/3 52 ± 14 35 ± 18 7.7 ± 0.7 25 ± 22 40 ± 4

3 1/3 36 ± 3 5.5 ± 0.1 728 ± 872 97 ± 11

diabetic mutation carrier

nondiabetic mutation carrier

CONTROL

8 5/8 44 ± 10 36 ± 13 8.0 ± 1.3 40 ± 26 34 ± 14

8 1/8 14 ± 13

21 8/20 40 ± 11

5.6 ± 0.2 35 ± 22 56 ± 16

5.3 ± 0.2 436 ± 399 108 ± 18

a

Demographic details of patients and controls included in the current validation study and a previously published discovery study.3 Values are given as mean with standard deviations. Bicarbonate and amylase analysis was performed in secretin-stimulated duodenal juice as reported elsewhere.20,48

It has previously been shown that CEL-MODY patients have reduced levels of the digestive enzyme amylase in the secretinstimulated duodenal juice by the use of immunometric methods 20 as well as using mass spectrometry 3 and immunoblotting3 consistent with a reduced pancreatic function. Using SRM we here identified significantly (p < 0.05) and clearly reduced amylase levels in the secretin-stimulated duodenal juice of 13 CEL mutation carriers compared with 19 controls (Figure 1B). Furthermore, the SRM-determined amylase quantities of individual samples correlated significantly (R2 = 0.5496, P < 0.0001) with amylase enzyme activity levels using immunometric methods (Figure 1C). We also identified increased levels of trefoil factor 1 in the CEL mutation carriers as anticipated, in particular, for the patients with diabetes (Figure 4G). Furthermore, for the 20 most differentially increased proteins, we compared the fold change between CEL mutation carriers and controls from the previous discovery proteomics study to the fold change observed using SRM quantification. The corresponding pseudocorrelation plot (Figure 2) shows similar trends for all 20 proteins between both studies (i.e., increased levels of the same proteins in CEL mutation carriers). Taken together, the SRM assays seem to robustly quantify protein levels in secretin-stimulated duodenal juice.

Whitney U test were computed using Graph Pad prism (v. 6.0b, San Diego, CA) excluding null values. Principal component analysis (PCA) was performed using Stata 12.0 (Stata Statistical Software, Stata, College Station, TX) based on the z-score of every measured peptide quantity. Biomarker separation efficiency was evaluated using receiver operating characteristic (ROC) curves drawn point by point using the measured concentrations (empirical ROC) and using an interpolation obtained after modeling the different populations using nonsymmetrical normal distributions calibrated on the median and ±34 percentiles as described in ref 24. The corresponding areas under the curve (AUC) were obtained from the q values of the empirical data and by integrating the interpolated curve, respectively. Details of the implementation can be found in the compomics-utilities library.25

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RESULTS

Robust Protein Quantification in Secretin-Stimulated Duodenal Juice Using SRM Assays

We used targeted proteomics with SRM peptide assays to quantify protein levels in secretin-stimulated duodenal juice from eight nondiabetic and eight diabetic CEL mutation carriers in a large family with monogenic diabetes and exocrine dysfunction (CEL-MODY) as well as from 21 unrelated healthy controls (Figure 1A, Table 1). Furthermore, we defined advanced CEL-MODY disease by diabetes development (i.e., the diabetic CEL mutation carriers) in contrast with early disease (i.e., the nondiabetic CEL mutation carriers). The current study is a major extension of a previously published discovery proteomics study in the same MODY family,3 where we used the list of the most differentially expressed proteins in secretin-stimulated duodenal juice to design SRM assays for 20 proteins that were increased in three diabetic CEL mutation carriers (Table 2; Table 1 also provides demographic information for the previous study for comparison). To serve as a negative control, we also included the digestive enzyme amylase (previously shown to be decreased in CEL mutation carriers). As a positive control, we included trefoil factor 1, a protein known to be secreted from the diseased pancreas26 and identified in the previous study3 (increased in CEL mutation carriers although not at the level of the most differentially expressed proteins). We first constructed response curves to evaluate the linearity of the SRM assays (Supplementary Figure 1 in the Supporting Information). Supplementary Table 1 in the Supporting Information displays the signature peptides, transitions used for quantification, Q1 and Q3 m/z values for the endogenous and SIS peptides, and the collision energy used in the SRM analysis.

Disease Progression Monitoring in CEL Mutation Carriers

Figure 3A shows the repartition of all the 37 subjects in a PCA plot obtained using the 21 potential biomarkers. Nondiabetic CEL mutation carriers (blue; early disease) show the same trend as healthy controls (green). In contrast, diabetic CEL mutation carriers (red; advanced disease) are clearly separated from the two other groups. The candidate biomarkers thus seem to be accurate indicators of the diabetes status of the patient. Indeed, three SRM peptides could robustly (statistical AUC ≥ 0.90) diagnose earlier (nondiabetic) stages of CEL-MODY, examplified by elongation factor 1-alpha-1 (Figure 3B, Supplementary Figure 2 and Supplementary Table 2 in the Supporting Information). Nine SRM peptides (corresponding to eight proteins and examplified by the proteins glutamate dehydrogenase 1, trefoil factor 1, pyruvate kinase isozymes M1/ M2, and 14-3-3 protein Sigma) could robustly diagnose (statistical AUC ≥ 0.90) advanced (diabetic) stages of CELMODY (Figure 3C−F, Supplementary Figure 3 and Supplementary Table 2 in the Supporting Information). One Group of SRM Peptides Associated with Early and One Group Associated with Advanced CEL-MODY Disease

We identified two groups of SRM peptides when we assessed a potential relationship between the SRM peptide quantities and 524



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Table 2. Overview of Peptides Included in the SRM Assay, Including Peptides That Failed during Optimization accession number

peptide(s)

status

14-3-3 sigma

protein

P31947

6-phosphogluconate dehydrogenase

P52209

60S acidic ribosomal protein P0

P05388

60S acidic ribosomal protein P1 60S acidic ribosomal protein P2

P05386 P05387

alpha-amylase 2B

P19961

band 4.1-like protein 3

Q9Y2J2

cadherin-related family member 2

Q9BYE9

chloride intracellular channel protein 1

O00299

chromogranin-A

P10645

cystatin-C

P01034

elongation factor 1-alpha 1

P68104

epididymal secretory protein E1

P61916

fructose-bisphosphate aldolase B

P05062

glutamate dehydrogenase 1

P00367

inorganic pyrophosphatase

Q15181

isocitrate dehydrogenase

O75874

keratin, type I cytoskeletal 18

P05783

keratin, type II cytoskeletal 8

P05787

pyruvate kinase isozymes M1/M2

P14618

tetranectin

P05452

YLAEVATGDDK VETELQGVCDTVLGLLDSHLIK GEELSCEER GILFVGSGVSGGEEGAR VGTGEPCCDWVGDEGAGHFVK TELDSFLIEITANILK AGAIAPCEVTVPAQNTGLGPEK VLALSVETDYTFPLAEK GHLENNPALEK AAGVNVEPFWPGLFAK NIEDVIAQGIGK ILDSVGIEADDDR YVASYLLAALGGNSSPSAK TGSGDIENYNDATQVR TSIVHLFEWR GLPAGTYCDVISGDK AVLEQEETAAASR TETISFGSVSPGGVK VILLDGSEYTCDVEK EAIDPALEGR DDDSGNNGVILFS DDDSGNNGVILFSILR DVNDNPPTLDVASLR GVTFNVTTVDTK LCPGGQLPFLLYGTEVHTDTNK NSNPALNDNLEK ELQDLALQGAK YPGPQAEGDSEGLSQGLVDR ALDFAVGEYNK EDSLEAGLPLQVR ALDFAVGEYNK LVGGPMDASVEEEGVR TQPNLDNCPFHDQPHLK IGGIGTVPVGR YYVTIIDAPGHR EVNVSPCPTQPCQLSK DCGSVDGVIK SGINCPIQK ELSEIAQSIVANGK LDQGGAPLAGTNK ETTIQGLDGLSER IIAEGANGPTTPEADK YSTDVSVDEVK HGGTIPIVPTAEFQDR AAPFSLEYR GQYISPFHDIPIYADK VEITYTPSDGTQK VEITYTPSDGTQK DIFQEIYDK AQIFANTVDNAR LEAEIATYR ASLENSLR VIDDTNITR ASLEAAIADAEQR YEELQSLAGK LQAEIEGLK GDYPLEAVR APIIAVTR GVNLPGAAVDLPAVSEK EQQALQTVCLK LDTLAQEVALLK

included failed failed included failed failed included failed failed included included failed failed included included failed included failed failed included failed failed failed included failed failed included failed failed failed included included failed included failed included failed failed included failed failed included included failed included failed included failed failed included included failed failed included included included included failed failed included included

525

gene name SFN

PGD

RPLP0

RPLP1 RPLP2

AMY2B

EPB41L3

CDHR2

CLIC1

CHGA

CST3

EEF1A1 NPC2

ALDOB

GLUD1

PPA1 IDH1

KRT18

KRT8

PKM2

CLEC3B



Article

Table 2. continued protein

trefoil factor 1

accession number

P04155

peptide(s)

status

TFHEASEDCISR TENCAVLSGAANGK QNCGFPGVTPSQCANK GCCFDDTVR

failed failed included failed

gene name

TFF1

few quantitative proteomics studies of pancreatic29−32 and secretin-stimulated duodenal fluid3,33 and, to our knowledge, none using SRM assays. Here we observed a robust correlation between SRM quantities of amylase and the corresponding enzymatic level of the enzyme for a high dynamic range of amylase levels in secretin-stimulated duodenal juice from healthy and diseased subjects, and we also confirmed previous findings of increased levels of trefoil factor 1 in patients with a diseased pancreas,26 both supporting the usefulness of the SRM assay in a new clinical setting. SRM moreover presents the huge advantage to monitor the abundance of multiple proteins, eventually targeting entire pathways, in a single measurement.27 The combination of evolving clinical phenotypes (such as distinct disease stages) and correlated protein biomarker levels in body fluids may increase the screening efficiency for severe pancreatic disease.34 CEL-MODY patients display distinct disease stages where, in particular, diabetes development is clearly correlated with the advancement of a more severe pancreatic disease with the simultaneous development of clinical signs of pancreatic exocrine dysfunction1 of pancreatic cysts3 and of MAPK-related proteins and cytokines.3 In the current study, we validated several potential diagnostic markers, including at least one marker of the positive mutation status (elongation factor 1-alpha 1) and at least four markers of the diabetes development (glutamate dehydrogenase 1, trefoil factor 1, 14-3-3 sigma, and pyruvate kinase isozymes M1/ M2). Given the increased risk of pancreatic cancer in patients with pancreatic cysts,35 further studies are warranted, assessing the levels of these markers in secretin-stimulated duodenal juice from patient cohorts with various forms of pancreatic cystic disease. We identified one group of SRM peptides associated with early CEL-MODY disease and one group of SRM peptides associated with advanced CEL-MODY disease. We found four SRM peptides (corresponding to the four proteins: elongation factor 1-alpha 1, keratin 8, epidiymal secretory protein 1, and 14−3−3 sigma) associated with early CEL-MODY disease compared with only three SRM peptides (including the protein elongation factor 1-alpha 1) using the ROC AUC (for markers of positive mutation status), probably due to the more stringent cutoff threshold (statistical AUC ≥ 0.90). Indeed, the difference in protein levels between nondiabetic CEL mutation carriers and controls was more significant for elongation factor 1- alpha 1 (p < 0.0001) than for the other three proteins (keratin 8, p < 0.01; epidiymal secretory protein 1, p < 0.01); 14−3−3 sigma, p < 0.05). Among the proteins corresponding to SRM peptides associated with early CEL-MODY disease, keratin 8 is (together with keratin 18) a major component of the intermediate filament cytoskeleton of pancreatic acinar cells and plays a relevant role in pancreatic exocrine homeostasis. Overexpression of keratin 8 leads to acinar changes and adipose conversion in mice.36 The level of keratin 8 has also been found to increase nearly three-fold upon pancreas injury and have been suggested to have a protective role.37 Epididymal

Figure 2. Pseudocorrelation plot of the proteins quantified with TMT (black line) and SRM (gray line) expressed as log2 (diabetic CEL mutation carriers carriers/healthy controls). TMT n = 6 (3 healthy controls + 3 diabetic CEL mutation carriers). SRM n = 29 (21 healthy controls + 8 diabetic CEL mutation carriers).

the early and advanced disease stages of CEL-MODY (Figure 4, Supplementary Figure 4 in the Supporting Information). The first group (Figure 4A−D; SRM peptides corresponding to the proteins keratin 8, elongation factor 1-alpha 1, epididymal secretory protein E1, and 14-3-3 sigma) showed significant increased quantities both in nondiabetic CEL mutation carriers compared with healthy controls and in diabetic CEL mutation carriers compared with nondiabetic CEL mutation carriers and suggests markers that may be important both for early and advanced disease stages of CEL-MODY pancreatic disease progression. The second group (Figure 4E−G; SRM peptides corresponding to glutamate dehydrogenase, pyruvate kinase isozymes M1/M2, and trefoil factor 1) showed significant increased quantities only in diabetic CEL mutation carriers compared with nondiabetic CEL mutation carriers and suggests markers that may be important for the development of the advanced stage (diabetic) of CEL-MODY pancreatic disease.

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DISCUSSION SRM assays have not yet become a part of the clinical routine; however, in a large interlaboratory study sponsored by the U.S. National Cancer Institute (NCI), performed in 2009, the reproducibility of SRM was demonstrated, thus paving the way to the clinic.12 SRM has been used to validate quantitative proteomics findings in a wide range of cellular experiments and body fluids.27 Although pancreatic fluid and secretin-stimulated duodenal juice may potentially constitute promising proximal fluids4,28 for studies of the diseased pancreas, there are only a 526



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Figure 3. (A) Principal component analysis (PCA) of all 37 patients based on the SRM-based quantities of 21 biomarker candidates. ROC curves (dotted line for empiral ROC curves and diamonds for statistical ROC curves) of individual proteins with statistical AUC ≥ 0.90 when comparing nondiabetic CEL mutation carriers from healthy controls for (B) elongation factor 1-alpha 1 (IGGIGTVPVGR) and diabetic CEL mutation carriers from nondiabetic CEL mutation carriers for (C) trefoil factor 1 (QNCGFPGVTPSQCANK), (D) glutamate dehydrogenease 1 (IIAEGANGPTTPEADK), (E) 14-3-3 protein sigma (YLAEVATGDDK), and (F) pyruvate kinase isoenzymes M1/M2 (GDYPLEAVR).

proteins known to be associated with diabetes and are listed in the Human Diabetes Proteome Project (HDPP),43 and pyruvate kinase isozymes M1/M2 has been associated with the Warburg effect in pancreatic cancer lines44 as well as being listed as a potential urine biomarker of renal cancer disease.16 These two proteins are both present in moderate levels in pancreatic beta cells (Protein Atlas45). Trefoil factor 1 is a secretory polypeptide, which is highly abundant in gastric and dudenal glandular cells and is believed to stabilize the mucosa layer.46 Very low levels are expressed (at the mRNA level) in

aecretory protein 1 is associated with a lysosomal storage disease (Niemann−Pick disease, type C2), where the protein is involved in the movement of lipids from late endosomes or lysosomes.38 Elongation factor 1-alpha 1 is involved in protein translation39 and 14-3-3 sigma promotes cell survival and is associated with several cancer types by interacting with p53,40 including pancreatic adenocarcinoma.41,42 Among the proteins corresponding to SRM peptides associated with advanced CEL-MODY disease, glutamate dehydrogenase 1 and pyruvate kinase isozymes M1/M2 are 527



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Figure 4. Scatter plot of individual peptides plotted as median with interquartile range for: (A) keratin, type I cytoskeletal 8 (YEELQSLAGK), (B) elongation factor 1-alpha 1 (IGGIGTVPVGR), (C) epididymal secretory protein E1 (EVNVSPCPTQPCQLSK), (D) 14-3-3 protein sigma (YLAEVATGDDK), (E) glutamate dehydrogenease 1 (IIAEGANGPTTPEADK), (F) pyruvate kinase isoenzymes M1/M2 (GDYPLEAVR), and (G) trefoil factor 1 (QNCGFPGVTPSQCANK). * indicates p value 0.01 to 0.05, ** indicates p value 0.001 to 0.01, *** indicates p value

Carboxyl-Ester Lipase Maturity-Onset Diabetes of the Young Disease

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