1850 Weidong Zhou1 Michela Capello2,3 Claudia Fredolini1,2,3 Leda Racanicchi4 Lorenzo Piemonti4 Lance A. Liotta1 Francesco Novelli2,3 Emanuel F. Petricoin1 1 Center
for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA 2 Center for Experimental Research and Medical Studies, San Giovanni Battista Hospital, Turin, Italy 3 Department of Medicine and Experimental Oncology, University of Turin, Turin, Italy 4 Diabetes Research Institute, San Raffaele Scientific Institute, Milano, Italy
Received January 6, 2012 Revised February 7, 2012 Accepted February 27, 2012
Electrophoresis 2012, 33, 1850–1854
Short Communication
MS analysis reveals O-methylation of L-lactate dehydrogenase from pancreatic ductal adenocarcinoma cells L-lactate dehydrogenase (LDH) converts pyruvate to lactate when oxygen is absent or in short supply, and the enzyme plays a crucial role in cancer metabolism. The functions of many mammalian proteins are modulated by posttranslational modifications (PTMs), and it has been reported that LDH was subjected to several PTMs, including phosphorylation, acetylation, and methylation. In this present work, we characterized the PTMs of LDH from pancreatic ductal adenocarcinoma (PDAC) cells by electrophoresis and mass spectrometry, and identified 13 O-methylated residues from the enzyme. In addition, our qualitative analysis revealed differential methylation of LDH from normal duct cells. The preliminary findings from this study provide important biochemical information toward further understanding of the LDH modifications and their functional significance in pathophysiological processes of pancreatic cancer. Keywords: L-lactate dehydrogenase / Mass spectrometry / Metabolism / Methylation / Pancreatic ductal adenocarcinoma DOI 10.1002/elps.201200017
L-lactate dehydrogenase (LDH) converts pyruvate, the final product of glycolysis to lactate when oxygen is absent or in short supply, and it performs the reverse reaction during the Cori cycle in the liver [1]. The enzymes are homo or heterotetramers composed of M (LDH-A) and H (LDH-B) protein subunits encoded by the LDHA and LDHB genes, respectively: LDH-1 (4H) in the heart and red blood cells, LDH-2 (3H1M) in the reticuloendothelial system, LDH-3 (2H2M) in the lungs, LDH-4 (1H3M) in the kidneys, placenta and pancreas, and LDH-5 (4M) in the liver and striated muscle [2]. Using slices of living tissues, Otto Warburg studied the energy metabolism of a tumor and first reported that cancer cells produced large amounts of lactate even in aerobic condition [3]. Since then, the Warburg effect was observed in several tumors in which the cells predominantly produced energy by a high rate of glycolysis followed by lactate fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria like most normal cells, even in the presence of oxygen [4–6].
Correspondence: Dr. Weidong Zhou, Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd, MS 1A9, Manassas, VA 20110, USA E-mail:
[email protected] Fax: +1-703-993-4288
Abbreviations: LDH, L-lactate dehydrogenase; PDAC, pancreatic ductal adenocarcinoma
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It has been reported that human LDH was subjected to phosphorylation and acetylation [7, 8], and tyrosine phosphorylation of LDH-A was important for redox homeostasis in cancer cells [9]. Recently, using in vitro methylation and 2DE analysis of viral Src-transformed rat kidney epithelial cells, Chiou reported that LDH-B could be methylated; however, the exact position of the methylation was not known [10]. Overexpression of LDH-A or LDH-B subunit has been implicated in the pathogenesis and progression of many cancers [11–15], and LDH may constitute a valid therapeutic target for diseases [16, 17]. Our previous studies demonstrated that LDH-B was upregulated in pancreatic ductal adenocarcinoma (PDAC) [15]. PDAC is the fourth leading cause of cancer-related deaths in the United States and Europe. The absence of early symptoms or clinical-pathological markers results in diagnosis at a late, inoperable stage in more than 80% of cases. The mean life expectancy is 15–18 months for patients with local and regional disease, and only 3–6 months for those with metastatic disease [18–20]. Since posttranslational modifications (PTMs) regulate almost all aspects of cell life, and abnormal modification is a cause or consequence of cancers, knowing the PTMs of LDH can not only extend our knowledge of the biochemical characteristics of the enzyme, but also offer opportunities for targeting the enzyme in cancer cells. Therefore, we exploited 1DE and LCMS/MS to examine the PTMs of LDH from CFPAC-1 cells
Colour Online: See the article online to view Figs. 1 – 3 in colour.
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General
Electrophoresis 2012, 33, 1850–1854
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Table 1. A list of the O-methylated peptides identified by LTQ-Orbitrap
Protein and peptides
Positiona)
LDH-A DLADE#LALVDVIEDK DLADELALVDVIE#DK VIGSGCNLD#SAR
43−57 43−57 158−169
QVVE#SAYEVIK
233−243
QVVESAYE#VIK
233−243
VTLTSE#EEAR
306−315
VTLTSEEE#AR LDH-B
306−315
LIAPVAEE#EATVPNNK
8−23
LIAPVAEEE#ATVPNNK
8−23
SLADE#LALVDVLEDK
44−58
SLADELALVDVLE#DK
44−58
M*VVE#SAYEVIK
234−244
LKDDE#VAQLK
309−318
Normal duct √
√
√
√
√
√
√
CFPAC-1 √
√
√
√
√
√
√
√
√
√
√
√
√
a) Position of the initial and final peptide amino acids in the protein sequence.
Figure 1. SDS-PAGE of cell lysates from CFPAC-1 and normal duct cells. A total of 50 g of proteins from each sample were loaded to the wells of the Novex 4–20% Tris-Glycine Gel (Invitrogen) to separate the proteins by SDS-PAGE. Protein bands corresponding to LDH were excised from the gel and in-gel digested with trypsin for MS analysis.
(metastatic cell line derived from PDAC patients, ECACC reference number: 91112501) and normal pancreatic duct cells. The advantages of cell lines were their easy accessibility and homogeneity, whereas pancreatic cancer tissue specimen was heterogeneous, containing high percentage of stromal cells, and the yield of duct cells from primary culture of pancreatic cancer tissue was low. CFPAC-1 cells were cultured at 37⬚C in DMEM (Invitrogen; Carlsbad, CA, USA) supplemented with 20 mM glutamine, 10% fetal calf serum (FCS), and 40 g/mL Gentamycin with humidified 5% CO2 . The cells were harvested and washed with Hank’s balanced salt solution (SigmaAldrich; Saint Louis, MO, USA). The cell pellet was freezedried overnight and stored at −80⬚C until use. Normal human pancreatic duct cells were obtained by primary culture of pancreatic duct from a single brain death donor under IRB approval (San Raffaele Scientific Institute; Milano, Italy). The duct cells were cultured in medium DMEM/F12 (1:1), supplemented with 2mM glutamine, 10% FCS, 100 U/mL penicillin, and 100 g/mL streptomycin. Through a period of C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
suspension culture, epithelial cells were enriched while stromal components were reduced to less than 1%, confirmed by FACS analysis with markers for epithelial (ESA, Ca19.9) and fibroblast (CD73, CD105, CD90) phenotype. The CFPAC1 and normal duct cells were resuspended for 1 h in lysis buffer consisting of Tris/HCl (50mM, pH 7.4), NaCl (150 mM), Triton X-100 (0.5% w/v), NP-40 (0.5% w/v), 80 mM DTT, 10 L/mL protease inhibitor cocktails (Sigma-Aldrich), 1 mM PMSF, 1 mM Na3 VO4 , and PhosStop phosphatase inhibitor cocktail (Roche Applied Science; Indianapolis, IN, USA), sonicated for 30 s, and centrifuged at 16 000 × g for 10 min. The protein concentration of supernatant was measured by Bradford Assay (BioRad; Hercules, CA, USA). The supernatants were then precipitated with 4 volume of acetone (Sigma-Aldrich) overnight at −20⬚C and centrifuged at 9000 × g for 5 min. The pellets were dried by lyophilization (Heto Drywinner; Birkerod, Denmark) for 2 h. The cell pellets were resuspended in SDS-PAGE loading buffer. BenchMark prestained protein ladder (Invitrogen) and 50 g of proteins from each sample were loaded to the wells of the Novex 4–20% Tris-Glycine Gel (Invitrogen) to separate the proteins by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R-250, allowing visualization of the separated proteins (Fig. 1). We cut the gel bands that have molecular weight approximately 37 kDa, corresponding to monomer LDH-A (36.7 KDa) and LDH-B (36.6 KDa), and the proteins in the bands were in-gel digested [21]. The extracted tryptic peptides were analyzed by high sensitive nanospray LCMS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific; Waltham, MA, USA). LC-MS/MS has been used routinely for characterization of PTMs from complex biological mixtures [22–24], and the LTQ-Orbitrap provides high accuracy mass measurement that is essential for the www.electrophoresis-journal.com
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Electrophoresis 2012, 33, 1850–1854
Figure 2. Identification of O-methylation of LDH-A and LDH-B by LC-MS/MS. (A) Example CID spectrum of the identified peptide DLADE#LALVDVIEDK (2+ ion m/z 836.4365) of LDH-A from normal duct cells; (B) CID spectrum of the identified peptide QVVESAYE#VIK (2+ ion m/z 639.8483) of LDH-A from CFPAC-1; (C) CID spectrum of the identified peptide M*VVE#SAYEVIK (2+ ion m/z 649.3382 with methionine oxidation) of LDH-B from CFPAC-1. The spectrum (left panel) is labeled to show b ions, y ions, and neutral loss of water from parent ions. The right panel is the table of the fragment assignments of the peptide, in which the matched b ions are colored with red, y ions with blue, and ions containing modified residue with cyan.
validation of modified peptide identifications and the reduction of false-positive identifications. The reversed-phase LC column was slurry-packed in-house with 5 m, 200 A˚ pore size C18 resin (Michrom BioResources, CA, USA) in a 100 m id × 10 cm long piece of fused-silica capillary (Polymicro Technologies, Phoenix, AZ, USA) with a laser-pulled tip. After sample injection, the column was washed for 5 min with mobile phase A (0.1% formic acid), and peptides were eluted using a linear gradient of 0% mobile phase B (0.1% formic acid, 80% ACN) to 50% B in 120 min at 200 nL/min, then to 100% B in an additional 5 min. The LTQ-Orbitrap was operated in a data-dependent mode in which each full MS scan (60 000 resolving power) was followed by eight MS/MS scans where the eight most abundant molecular ions were dynamically selected and fragmented by CID using a normalized collision energy of 35%. The “FT master scan preview mode”, “Charge state screening”, “Monoisotopic precursor selection”, and “Charge state rejection” were enabled so that only the 1+, 2+, and 3+ ions were selected and fragmented by CID. Tandem mass spectra collected by Xcalibur (version 2.0.2) were searched against the NCBI human protein database using SEQUEST (Bioworks software from Thermo Fisher, version 3.3.1) with full tryptic cleavage constraints, static cysteine alkylation by iodoacetamide, and variable me-
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thionine oxidation. Mass tolerance for precursor ions was 5 ppm and mass tolerance for fragment ions was 0.25 Da. The SEQUEST search results were filtered by the criteria “Xcorr versus charge 1.9, 2.2, 3.0 for 1+, 2+, 3+ ions; ⌬Cn >0.1; probability of randomized identification of peptide
