1. 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 represent
a key participant to glucose homeostasis. Insulin is packed and processed within
these vesicles before its release by exocytosis. It is therefore vital to acquire qualitative
and quantitative data on the ISG proteome, in order to increase our knowledge on ISG
biogenesis, maturation and exocytosis. Many efforts have been made in the past years to
increase the coverage of the ISG proteome. However, much of the existing work on the
composition of granules was done in immortalized cell lines or rodent tissues which may not
faithfully reflect the composition of granules in human islets. The major goal of the
current research was to characterize the proteome of an enriched fraction of ISGs from
the human beta cells. Herein, we present the global proteomic profiling of human ISGs
using two analytical methodologies coupled with nanoliquid chromatography-tandem mass
spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC ion trap-ESI-MS/MS), and
two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC-ESI-MS/
MS). The 253 significantly identified proteins (p<0.05) were further analyzed with
pathway analysis (IPA) to define the functional signature of the ISGs, which highlighted
the involvement of many proteins from the carbohydrate metabolism:
glycolysis/gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism
but also from the keto and amino acids metabolism (pyruvate, arginine, proline, methionine,
alanine, aspartate and the aromatic amino acids, among others). Proteins involved in
signaling pathways related to the oxidative stress and in the membrane fusion (such as
annexins 1,2,3 4, 5 and 7) were also identified in the proteome of ISGs. The elucidation
of the roles played by these proteins in the biogenesis and maturation of ISGs will further
help in the understanding of the mechanisms governing impaired insulin secretion during
diabetes.
INTRODUCTION RESULTS (I)
RESULTS (II)
Conclusions
Analysis of the mass spectrometry data revealed that the majority of the identified proteins
in the un-stimulated ISGs are, consistent with previous studies performed on rat islets,
involved in sugar metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway), protein
synthesis and degradation, cytoskeleton (re)-organization, and amino acids metabolism.
Upon stimulation of the ISGs with glucose a remarkable change in the ISGs proteome is
shown 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 a
better understanding of disease mechanisms, such as those that pertain to type 1 and type
2 diabetes mellitus, pancreatitis or pancreatic cancer (1-5). Importantly, studies on
specific cell types or organelles from the pancreas may better resolve proteomes upon which
comparative studies (e.g., diseased versus healthy, nascent versus mature) may then be
based.
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 resistance
results 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 and
granule contents can provide valuable information relevant to further understand metabolic
regulation 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 ISG
proteome. However, much of the existing work on the composition of granules was done in
immortalized cell lines or rodent tissues which may not faithfully reflect the composition of
granules in human islets.
The major goal of the current research was to characterize the proteome of an enriched
fraction of ISGs from the human beta cells. Herein, we present the global proteomic
profiling of human ISGs using two analytical methodologies coupled with nanoliquid
chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF
nanoLC Orbitrap-ESI-MS/MS), and two-dimensional fluorescence difference-in-gel
electrophoresis (2D-DIGE nanoLC-ESI-MS/MS).
•ISGs purification:
•Islets were isolated by enzymatic digestion and gradient purification from pancreata of multiorgan donors and cultured in
M199 medium containing 5.5 mmol/L glucose,supplemented with 10% (vol/vol) serum, 100 U/mL penicillin, 100μg/mL
streptomycin, 50μg/mL gentamicin, and 750 ng/mLamphotericin B (Sigma-Aldrich, St. Louis, MO) using similar methods
described elsewhere (1-5).
•1D-EF-proteomics: 1D-electrophoresis was carried on standard 12% SDS-PAGE gels using total protein extracts from the
stimulated and un-stimulated ISG purified secretory granules. Equals amounts of each sample were applied on individual wells
and the elctrophoresis was run at 120 V constant for 1 hour and 15 min. Bands were excised from the gel and further
subjected to in situ trypsin digest using standard protocol. The tryptic peptides were extracted and purified on zip-tip C18
and 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 plasma
and Cy5 for lymph) and to 2D-DIGE preparative electrophoresis (Applied BIOMICS Inc. facilities, Hayward, CA). After
electrophoresis, the gel was scanned using a Typhoon image scanner and the images were analyzed using the DeCyder
software. Protein spots of interest were automatically picked from the 2D gel with the Ettan Spot Picker, subject to tryptic
digestion 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_1Sept2007
database 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 a
fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 3.6 Da. Iodoacetamide derivative of cysteine was
specified in Mascot and X! Tandem as a fixed modification. Pyro-glu from E of the n-terminus, s-carbamoylmethylcysteine
cyclization (N-terminus) of the n-terminus, deamidated of unknown, deamidation of asparagine, hydroxylation of lysine and
oxidation 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 they
could 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 be
differentiated 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 during
exocytosis.
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 in
the 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 and
metabolical 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. Ratio
SG-S/
SG-O
1 361 -1.1
2 580 1.2
3 642 1.1
4 613 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),