Protein expression profile of human insulin secretory granules mapped by 2D-DIGE and 1D SDS-PAGE coupled with nanoLC-ESI-MS/MS bottom-up proteomics

Cristina C. Clement(1), Regina Kuliwat (2), Laura Santambrogio (1),(1) Pathology; and (2) Developmental & Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, 10461

ABSTRACT MATERIALS AND MEHODS

Insulin secretory granules (ISGs) are crucial organelles of pancreatic ß-cells and representa key participant to glucose homeostasis. Insulin is packed and processed withinthese vesicles before its release by exocytosis. It is therefore vital to acquire qualitativeand quantitative data on the ISG proteome, in order to increase our knowledge on ISGbiogenesis, maturation and exocytosis. Many efforts have been made in the past years toincrease the coverage of the ISG proteome. However, much of the existing work on thecomposition of granules was done in immortalized cell lines or rodent tissues which may notfaithfully reflect the composition of granules in human islets. The major goal of thecurrent research was to characterize the proteome of an enriched fraction of ISGs fromthe human beta cells. Herein, we present the global proteomic profiling of human ISGsusing two analytical methodologies coupled with nanoliquid chromatography-tandem massspectrometry: one-dimensional gel electrophoresis (1DEF nanoLC ion trap-ESI-MS/MS), andtwo-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC-ESI-MS/MS). The 253 significantly identified proteins (p<0.05) were further analyzed withpathway analysis (IPA) to define the functional signature of the ISGs, which highlightedthe involvement of many proteins from the carbohydrate metabolism:glycolysis/gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolismbut also from the keto and amino acids metabolism (pyruvate, arginine, proline, methionine,alanine, aspartate and the aromatic amino acids, among others). Proteins involved insignaling pathways related to the oxidative stress and in the membrane fusion (such asannexins 1,2,3 4, 5 and 7) were also identified in the proteome of ISGs. The elucidationof the roles played by these proteins in the biogenesis and maturation of ISGs will furtherhelp in the understanding of the mechanisms governing impaired insulin secretion duringdiabetes.

INTRODUCTION RESULTS (I)

RESULTS (II)

ConclusionsAnalysis of the mass spectrometry data revealed that the majority of the identified proteinsin the un-stimulated ISGs are, consistent with previous studies performed on rat islets,involved in sugar metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway), proteinsynthesis and degradation, cytoskeleton (re)-organization, and amino acids metabolism.Upon stimulation of the ISGs with glucose a remarkable change in the ISGs proteome isshown to be characterized by a shift towards the proteins networks involved in exocytosis,secretion and membrane fusion events.

Proteomic investigations of the pancreas have aimed to identify proteins that may provide abetter understanding of disease mechanisms, such as those that pertain to type 1 and type2 diabetes mellitus, pancreatitis or pancreatic cancer (1-5). Importantly, studies onspecific cell types or organelles from the pancreas may better resolve proteomes upon whichcomparative studies (e.g., diseased versus healthy, nascent versus mature) may then bebased.The major islet beta-cell secretory product, insulin, is crucial to metabolic homeostasis,and stimulates the anabolic processes of glucose uptake, lipid synthesis and deposition,protein synthesis and growth while simultaneously suppressing catabolism of glycogen,triacylglycerol and protein. Dysfunctional production combined with insulin resistanceresults in dysregulation of glucose and lipid metabolism and type-2 diabetes mellitus (T2DM)(2-4). As a result, proteomics methods to analyze beta-cell secretion mechanisms andgranule contents can provide valuable information relevant to further understand metabolicregulation and the etiology of diseases such as T2DM (1-4).Many efforts have been made in the past years to increase the coverage of the ISGproteome. However, much of the existing work on the composition of granules was done inimmortalized cell lines or rodent tissues which may not faithfully reflect the composition ofgranules in human islets.The major goal of the current research was to characterize the proteome of an enrichedfraction of ISGs from the human beta cells. Herein, we present the global proteomicprofiling of human ISGs using two analytical methodologies coupled with nanoliquidchromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEFnanoLC Orbitrap-ESI-MS/MS), and two-dimensional fluorescence difference-in-gelelectrophoresis (2D-DIGE nanoLC-ESI-MS/MS).

•ISGs purification:

•Islets were isolated by enzymatic digestion and gradient purification from pancreata of multiorgan donors and cultured inM199 medium containing 5.5 mmol/L glucose,supplemented with 10% (vol/vol) serum, 100 U/mL penicillin, 100μg/mLstreptomycin, 50μg/mL gentamicin, and 750 ng/mLamphotericin B (Sigma-Aldrich, St. Louis, MO) using similar methodsdescribed elsewhere (1-5).

•1D-EF-proteomics: 1D-electrophoresis was carried on standard 12% SDS-PAGE gels using total protein extracts from thestimulated and un-stimulated ISG purified secretory granules. Equals amounts of each sample were applied on individual wellsand the elctrophoresis was run at 120 V constant for 1 hour and 15 min. Bands were excised from the gel and furthersubjected to in situ trypsin digest using standard protocol. The tryptic peptides were extracted and purified on zip-tip C18and further subjected to LTQ-MS/MS sequencing.

•2D-EF-proteomic: 2D DIGE Protein Expression Profiling of protein extracts from the glucose-stimulated and un-stimulated secretory granules

•Equal amounts of protein from each protein extract were subjected to fluorescence labeling using 2 dyes (Cy3 for plasmaand Cy5 for lymph) and to 2D-DIGE preparative electrophoresis (Applied BIOMICS Inc. facilities, Hayward, CA). Afterelectrophoresis, the gel was scanned using a Typhoon image scanner and the images were analyzed using the DeCydersoftware. Protein spots of interest were automatically picked from the 2D gel with the Ettan Spot Picker, subject to trypticdigestion and MS/MS analysis.

DATABASE SEARCHING. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version Mascot)and X! Tandem (The GPM, thegpm.org; version 2007.01.01.1). X! Tandem was set up to search a subset of the nr_1Sept2007database also assuming trypsin. Mascot was set up to search the nr_1Sept2007 database (selected for Homo sapiens,unknown version, 194019 entries) assuming the digestion enzyme trypsin. Mascot and X! Tandem were searched with afragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 3.6 Da. Iodoacetamide derivative of cysteine wasspecified in Mascot and X! Tandem as a fixed modification. Pyro-glu from E of the n-terminus, s-carbamoylmethylcysteinecyclization (N-terminus) of the n-terminus, deamidated of unknown, deamidation of asparagine, hydroxylation of lysine andoxidation of methionine were specified in Mascot as variable modifications. Pyro-glu from E of the n-terminus, s-carbamoylmethylcysteine cyclization (N-terminus) of the n-terminus, deamidated of unknown, deamidation of glutamine,hydroxylation of proline and oxidation of methionine were specified in X! Tandem as variable modifications.

CRITERIA FOR PROTEIN IDENTIFICATION-- Scaffold (version Scaffold_2_06_00, Proteome Software Inc., Portland,OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if theycould be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm (Keller, A et al Anal.Chem. 2002;74(20):5383-92). Protein identifications were accepted if they could be established at greater than 95.0%probability and contained at least 1 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm(Nesvizhskii, AI Anal Chem. 2003 Sep 1;75(17):4646-58). Proteins that contained similar peptides and could not bedifferentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

Stimulated Un-Stimulated

O-un-stimulated ISGs.S-stimulated ISGs with glucose.

Figure 1: one-dimensional gel electrophoresis (1DEF nanoLC ion trap-ESI-MS/MS) analysis of O (unstimulated) vs. glucose-stimulated ISGs purified from human beta cells.

Stimulated

Un-Stimulated Stimulated Un-Stimulated Stimulated Un-Stimulated

Figure 3: one-dimensional gel electrophoresis (1DEF nanoLC ion trap-ESIMS/MS) analysis of O(unstimulated) vs. glucose-stimulated ISGs purified from human beta cells reveals the down-regulation of many annexins proteins, a process mostly due to the membrane fusion events duringexocytosis.

Figure 4: one-dimensional gel electrophoresis (1DEF nanoLC ion trap-ESIMS/MS) analysis of O(unstimulated) vs. glucose-stimulated ISGs purified from human beta cells: major hits identified inthe two sample sets using the Scaffold viewer and the protein probabilities scoring functions.

Figure 5: one-dimensional gel electrophoresis (1DEF nanoLC ion trap-ESIMS/MS) analysis of O (unstimulated) vs. glucose-stimulated ISGs purified from human beta cells: the functional andmetabolical pathways analysis using two independent engines (IPA and David Bioinformatics) reveals enzymes and proteins associated with the sugar and amino acids metabolism in the un-stimulated ISGs.

Protein ID Spot No. RatioSG-S/ SG-O

1 361 -1.12 580 1.23 642 1.14 613 1.7

5 637 2.1

GAPDH

(glyceraldehyde

dehydrogenase)

Figure 6: 2D-EF-proteomic: 2D DIGE Protein Expression Profiling of protein extracts from the glucose-stimulated (S) and un-stimulated secretory granules(O),