Proteomic Analysis of Microsomes from Lactating Bovine Mammary Gland Lifeng Peng,† Pisana Rawson,† Danyl McLauchlan,† Klaus Lehnert,‡ Russell Snell,‡ and T. William Jordan*,† Centre for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand, and ViaLactia Biosciences, Auckland, New Zealand Received December 5, 2007
Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESIMS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue. Keywords: Lactation • lipids • mammary • metabolism • proteome • secretion • synthesis
Introduction Our aim is to characterize the proteome of mammary tissue that is involved in production of lipids. Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. At the onset of lactation there is substantial morphological and metabolic remodeling of mammary alveolar epithelial cells for production of milk molecules.1 Although many of the soluble enzymes required for synthesis of fatty acids from glucose can be detected by two-dimensional electrophoresis (2-DE) of mammary tissue2–5 low-abundance and membrane proteins are poorly represented in these studies. Enzymes that are not observed using 2-DE of tissue homogenates include ER desaturases and acyltransferases required for modification of fatty acids and their ligation to glycerophosphate during synthesis of triacylglycerols.5 Centrifugal fractionation provides one approach to enrich organelles, including from mammary gland,6,7 and we have therefore used differential centrifugation to prepare a microsomal fraction for analysis. The microsomal fraction obtained after homogenization and differential centrifugation contains membrane-bound components including transport vesicles and vesicles produced by fragmentation of ER, Golgi, plasma membrane, lysosomes, mitochondria, peroxisomes, and other organelles during homogenization. ER is the site of several * Corresponding author: T. William Jordan, School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington 6140, New Zealand. Tel: +64 4 463 6092. Fax: +64 4 463 5331. E-mail:
[email protected]. † Victoria University of Wellington. ‡ ViaLactia Biosciences. 10.1021/pr700819b CCC: $40.75
2008 American Chemical Society
cellular processes including protein biosynthesis and transport, production of cholesterol, phospholipids, and triacylgycerols, in addition to fatty acid elongation, hydroxylation, and desaturation, and oxidation of xenobiotics and endogenous compounds. The Golgi complex is a major organelle of the secretory pathway, responsible for sorting, packaging, and distribution of newly synthesized proteins and membrane lipids as well as recycling molecules. In addition, some post-translational modifications of proteins including glycosylation are carried out by ER and Golgi enzymes. Moreover, microsomes contain vesicles whose proteins carry out essential cellular functions such as molecular trafficking and secretion. Microsomes are therefore a useful source for analysis of the proteome involved in milk production including synthesis and secretion of milk lipids. In the present study, we analyzed the proteome of an unfractionated microsomal pellet isolated from lactating bovine mammary tissue. Proteins were analyzed using one-dimensional SDS-PAGE (1-DE) coupled with RPLC-ESI-MS/MS. We identified 703 proteins, of which 52 were directly associated with lipid metabolism, transport, and secretion. Some of these proteins have not previously been reported from proteomics analyses of mammary tissue
Materials and Methods Sample Preparation. Udders were obtained from lactating Friesian dairy cows at the Wellington abattoir. Pieces of mammary tissue (ca. 1 × 2 × 4 cm) were excised from the center of the left front quarter, avoiding tissue from the cisternal region, and were perfused with ice-cold 5 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, and 1% protease inhibitor Journal of Proteome Research 2008, 7, 1427–1432 1427 Published on Web 03/07/2008
research articles
Peng et al.
Figure 2. Subcellular localizations of proteins in the microsomal fraction.
Figure 1. 1-D SDS-PAGE of the microsomal fraction. Lane a, microsomal sample; lane b, protein markers.
cocktail containing 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatinA, E-64, bestatin, leupeptin, and aprotinin (SigmaAldrich, catalog number P8340). The tissue was further minced into small pieces (0.3–0.5 cm2), washed twice with the above medium, and blotted on a paper towel. Approximately 5 g of tissue was suspended in 20 mL of the medium and homogenized on ice using a Kinematica Polytron homogenizer with a PT-20 probe at speed 5 for 40–60 s using 10 s intervals. The suspension was centrifuged at 800g for 10 min to sediment nuclei and cell debris, and the supernatant was centrifuged at 10 000g for 15 min. The resulting supernatant was centrifuged at 105 000g for 1 h to produce a microsomal pellet that was washed twice by resuspension in fresh medium and centrifugation at 105 000g for 1 h. The centrifugation was at 4 °C. The final centrifugal pellet was resuspended in sample buffer containing 40 mM Tris, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (w/v) aminosulfobetaine-14, and protease inhibitors as above for proteomic analysis. Protein concentrations were determined using a 2-D Quant Kit (GE Healthcare). One-Dimensional SDS-Polyacrylamide Gel Electrophoresis. Microsomal pellets dissolved in the sample buffer were mixed with Invitrogen NuPAGE lithium dodecyl sulfate sample buffer and aliquots (40 µg protein per lane) were electrophoresed on 4–12% precast polyacrylamide Bis-Tris NuPage gels using MOPS SDS electrophoresis buffer with addition of 0.5 mL of antioxidant (Invitrogen, Carlsbad, CA) in the upper chamber. After electrophoresis, the gels were stained using colloidal Coomassie brilliant blue G-250 and scanned using a Molecular Dynamics Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) as previously described.5 Nanoflow RPLC-ESI-MS/MS. 1-DE gel lanes were sliced into 37 bands, and the gel bands were diced into cubes 1 × 1 mm2. The sliced and diced gel pieces were destained in 50% ACN in 50 mM ammonium bicarbonate, reduced with 10 mM DTT in 0.1 M NH4HCO3, incubated for 30 min at 56 °C, and alkylated with 55 mM iodoacetamide in 0.1 M NH4HCO3 for 20 min at 1428
Journal of Proteome Research • Vol. 7, No. 4, 2008
room temperature in the dark. The reduced and alkylated proteins were then in-gel-digested with 0.25 µg of trypsin (Roche modified sequencing grade) in 50 mM ammonium bicarbonate at 37 °C overnight. The tryptic peptides were sequentially extracted with 10 µL of 25 mM NH4HCO3, 10 µL of ACN, and 50 µL of 5% formic acid, and then 10 µL of ACN again for 15 min at 37 °C with shaking at each step. The extracts were pooled and dried under vacuum to approximately 5 µL and then dissolved in 0.1% formic acid in 2% ACN in water. RPLC-ESI-MS was carried out using a Dionex UltiMate 3000 LC system (LC Packings, Netherlands) and a Thermo Finnigan LTQ mass spectrometer equipped with a nanospray ion source (Thermo Electron Corporation). Peptides were separated on a 75 µm × 15 cm PepMap C18 analytical column (3 µm, 300 Å Dionex) at a constant flow rate of 200 nL/min using a gradient constructed from 0.1% formic acid (solvent A) and 0.1% formic acid in 98% ACN (solvent B): 2% B for 5 min; 2–25% B for 45 min; 25–50% B for 20 min; 50–95% B for 5 min; 95% B for 5 min. The LTQ was operated in data-dependent tandem MS mode where the five most abundant precursor ions detected in a single MS scan from m/z 400 to m/z 2000 were dynamically selected for subsequent MS MS scans with collision energy set to 35%, simultaneously incorporating dynamic exclusion option to prevent reacquisition of MS/MS spectra of the same peptides. MS/MS Data Analysis. Acquired MS and MS/MS data were interpreted using the SEQUEST algorithm (Version 3.2 SR2, Thermo Finnigan). The database used was a composite of the bovine subset of the NCBInr (38 045 protein entries, 09 October 2006) and the reverse database created by reversing the order of the amino acid sequence for each protein to allow estimation of false-positive identifications. Spectra were searched with peptide mass tolerance of 2 Da, fragment ion tolerance of 0.8 Da allowing variable modifications of methionine oxidation and cysteine carbamidomethylation, and a maximum of 2 missed internal full trypsin cleavage. Results were filtered using the criteria of Xcorr g 1.8 for +1 charge state, g 2.7 for +2 charge state, and g 3.6 for +3 charge state, ∆Cn cutoff value g 0.1 and protein probability e 1 .00 × 10-3. Any peptide passing the filter parameters that was derived from the reverse database was defined as false-positive. The false-positive discovery rate was estimated according to Peng et al.,10 which was controlled less than 1%. If the peptide sequence was uniquely matched to a protein, the peptide was classified as a unique peptide. Proteins with 2 or more unique peptides were considered positive identifications. When a protein was identified by a single unique peptide, the corresponding MS/MS spectrum was further manually inspected and validated as described by Chen et al.11 Some filtered peptide sequences were matched to
Microsomal Proteome
research articles
Figure 3. Biological function classification of microsomal proteins.
multiple proteins due to sequence homologies. In these cases, proteins matched to the same peptide sequences were allocated to protein groups. Within each protein group, proteins with more than 3 peptide matches were considered a positive identification, and only a single protein from the group with either the largest sequence coverage or with representative annotation was reported.12 In addition, the MS/MS data set was analyzed using Mascot (Version 2.1.03. Matrix Science, London, U.K.) against the same bovine NCBInr database again allowing variable modification of methionine oxidation, cysteine carbamidomethylation, and a maximum of 2 missed internal full trypsin cleavages as a complementary approach to the SEQUEST searches.13 Criteria for a match to the sequence database were at least 2 matched peptides whose individual Mascot scores were at least 20. Classification of Proteins. Identified proteins were classified according to their subcellular localization and biological function using GOst (http://www.godatabase.org/cgi-bin/gost/gost.cgi) and PSORT II prediction (http://psort.nibb.ac.jp/ form2.html). Proteins with no annotated gene ontology were classified manually according to literature searches. Transmembrane helices in proteins were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html).
Results and Discussion Protein Profiling of the Microsomal Fraction. The microsomal pellet was prepared by differential centrifugation of a homogenate of mammary tissue. With this protocol, proteomes of membrane fractions have been reported for various biological cells and tissues. As the microsomal fraction predominantly consists of membrane-bound particles derived from ER and other organelles, 0.5% aminosulfobetaine-14 was included in the sample preparation buffer to aid solubilization of membrane proteins.14 Aliquots of the microsomal sample were electrophoresed on an SDS-PAGE gel (Figure 1). Thirty-seven slices were cut from the gel and subjected to digestion with trypsin. Duplicate aliquots of the digest were analyzed by nanoflow RPLC-ESI-MS/MS. The MS/MS spectra were searched against the composite bovine database using the SEQUEST and Mascot algorithms with false-positive rate less than 1%. Manual validation of spectra was carried out for matches based on a single peptide. The SEQUEST and Mascot results were combined to generate a protein list. This approach led to 703 protein identifications. SEQUEST and Mascot searches pro-
duced overlapping but different protein lists as is wellestablished for these complementary approaches for analysis of tandem mass spectra.13,15 Annotations for 667 of these proteins were available (Supplementary Table S1 in Supporting Information), 36 were unknown or hypothetical. These unknown or hypothetical proteins represent a source of candidates to explore novel functions with further potential to assign these proteins to organelles. We therefore performed conserved domain-search in the conserved domain database in NCBI for the unknown and hypothetical protein sequences (Supplementary Table S2 in Supporting Information). Among the hypothetical proteins for which function was predicted, we located a peroxisomal delta(3), delta(2) enoyl-CoA isomerase that is associated with isomerization of double bonds in fatty acids. Figure 2 shows a classification of the subcellular localizations of the 703 proteins identified in the microsomal fraction by the 1-DE approach. Biological function categories of proteins from ER, Golgi, lysosomes, peroxisomes, and plasma membrane are shown in Figure 3. Processes associated with protein synthesis, sorting, processing, and transport accounted for over three-quarters of the identifications. The next largest category was proteins involved in lipid metabolism transport and secretion. These identifications are consistent with the importance of protein and lipid synthesis and secretion in lactating mammary gland. Golgi proteins included a β-galactosyltransferase (lactose synthase) that was not detected by 2-DE of mammary homogenates.5 Annotation as plasma membrane origin was found for 188 of the 703 identified proteins, consistent with sedimentation of plasma membrane derived vesicles in the microsomal fraction. One hundred and sixty of the total 703 proteins had predicted transmembrane domains (Supplementary Table S1 in Supporting Information). Although we are cautious about some of the annotations and about prediction of transmembrane domains, these results confirm the prevalence of membrane proteins in the microsomal fraction. Furthermore, a number of proteins presumed to be of moderate to low abundance, including membrane receptors (e.g., inositol 1,4,5-trisphosphate receptor, mannose-6-phosphate receptor), transporters (e.g., chloride channel and solute carrier subunits), and signal transduction components (e.g., c-GMP-stimulated phosphodiesterase), were identified. The microsomal fraction contained a number of proteins from the cytoplasm, cytoskeleton, and nuclear sap (Figure 2). Possible explanations include dual membrane/extra-membrane localization, functional association of membranes with cytoJournal of Proteome Research • Vol. 7, No. 4, 2008 1429
research articles
Peng et al.
Table 1. Proteins Associated with Lipid Metabolism, Transport, and Secretion Identified in the Microsomal Fraction of Bovine Lactating Mammary Gland
protein name
2,4-Dienoyl-CoA reductase 3-Hydroxyacyl-CoA dehydrogenase type-2 Acetyl-CoA carboxylase Acyl-CoA dehydrogenase, very long chain Acyl-CoA synthetase long-chain family member 1 isoform 2 Adipocyte differentiation-related protein Adipocyte plasma membrane-associated protein Adipose differentiation-related protein-like Apolipoprotein A1 Apolipoprotein A-IV Apolipoprotein E Arachidonate 15-lipoxygenase
gi number
kDa/pI
number of peptides matched
1
β-Oxidation of fatty acids
83759185 27.12/8.43
4
β-Oxidation of short chain fatty acids
27806341 265.30/5.95 74267810 70.60/8.67
18 6
Cytoplasm; fatty acid synthesis β-Oxidation of fatty acids
76655661 81.64/8.24
17
27806759 49.37/8.72 74268319 46.06/6.65
13 7
Synthesis of long-chain fatty acyl-CoAs including palmitoyl-CoA Regulation of lipolysis Adipocyte differentiation
18032251 45.53/8.41
2
74268269 79160178 81 27806211
30.28/5.7 43.02/5.3 36.04/5.55 74.99/6.08
16 6 1 2
ATP citrate lyase Butyrophilin Cytochrome b5 Cytochrome P450 1A1 Cytochrome P450 2C24 Cytochrome P450 3A28 Cytochrome P450, family XIX Enoyl-CoA hydratase domain containing 1 Fatty acid binding protein, adipocyte-type Fatty acid binding protein, heart-type Fatty acid desaturase 2 Fatty acid synthase
79158721 2266432 1372997 30523216 76654714 5921920 27805827 88682928 895754 27805809 76657601 38425281
119.71/6.83 59.93/5.11 11.18/4.9 34.94/6.26 29.92/5.52 58.15/8.78 58.091/7.95 33.52/7.71 14.67/5.38 14.81/6.73 52.65/8.99 274.55/6.23
10 25 6 1 2 2 3 16 3 12 2 56
Fatty aldehyde dehydrogenase Hydroxy-delta-5-steroid dehydrogenase and isomerase Hydroxysteroid (11-beta) dehydrogenase 2 Hypothetical protein LOC505355 Lipoprotein lipase
76644087 54.04/7.53 27805923 42.09/8.04
2 2
27807157 44.0/8.55 77735439 23.5/9.54 163305 50.55/8.72
2 2 1
Low-density lipoprotein receptorrelated protein 1 precursor Low-density lipoprotein receptorrelated protein 1 precursor Lysophosphatidic acid acyltransferase Mammary gland factor STAT5B
76618579 121.24/5.07
4
76618581 248.93/5.18
12
14579219 32.00/9.55 5705869 88.44/5.71
3 1
Microsomal triglyceride transfer protein, large subunit precursor Milk fat globule-EGF factor 8 protein Monoglyceride lipase NADH-cytochrome b5 reductase
1709166 99.03/8.74
2
74354284 47.83/6.78 76649624 26.67/7.19 1709233 33.99/6.87
11 5 11
74268199 1322373 12643711 28411221
5 8 2 10
NADPH-cytochrome P450 reductase PAS-4 Phosphoinositide phospholipase C Phospholipid hydroperoxide glutathione peroxidase Progesterone membrane binding protein Prosaposin Prostaglandin F2 receptor negative regulator precursor Prostaglandin I2 synthase Retinol dehydrogenase 11 Serine palmitoyltransferase 2 Short chain 3-hydroxyacyl-CoA dehydrogenase Stearoyl-coenzyme A desaturase Sterol carrier protein 2 Vigilin Xanthine dehydrogenase
1430
biological function
1575000 35.64/9.21
77.22/5.38 53.31/8.39 117.12/6.32 19.93/8.3
Regulation of lipolysis Plasma lipoproteins Plasma lipoproteins Plasma lipoproteins Lipid metabolism, lipoxygenase activity, inflammatory response Synthesis of cytosolic acetyl-CoA Secretion of milk-fat droplets Electron carrier and sterol biosynthesis Oxidizes steroids, fatty acids and xenobiotics Metabolism of arachidonic acid Oxidizes steroids, fatty acids and xenobiotics Aromatase (conversion of androgen to estrogen) Fatty acid metabolism Regulation of fatty acid uptake and intracellular transport Regulation of fatty acid uptake and intracellular transport Delta(6) fatty acid desaturase Synthesis of long-chain fatty acids; insulin signaling pathway Oxidation of long-chain aliphatic aldehydes to fatty acids Steroid metabolism Steroid metabolism Metabolism of unsaturated fatty acids Metabolism of plasma lipoprotein triacylglycerols, delivery of fatty acids to tissues Cellular uptake of lipids Cellular uptake of lipids Triacylglycerol and phospholipid biosynthesis Signal transduction and transcription; mediate fatty acid synthesis by activation of acetyl-CoA carboxylase expression Transport of triacylglycerols, cholesteryl esters and phospholipids, secretion of plasma lipoproteins Lactadherin precursor, phosphatidylethanolamine binding Converts monoacylglycerols to free fatty acids and glycerol Desaturation and elongation of fatty acids, cholesterol biosynthesis, drug metabolism Reduction of P450s Fatty acid transport Phospholipid degradation; signal transduction Phospholipid metabolism
61859954 26.05/5.03 27806447 58.01/4.95 76612969 92.27/5.99
4 4 4
Steroid binding, steroid hormone receptor activity Lipid metabolism, sphingolipid metabolism Negative regulation of PGF2-alpha receptor
27806107 56.63/6.75 76628115 34.44/9.43
2 5
Prostacyclin biosynthesis Oxidoreduction of retinoids, metabolism of short-chain aldehydes Lipid metabolism, sphingolipid metabolism. β-Oxidation of short chain fatty acids Catalyzes the insertion of a double bond into fatty acyl-CoA Intracellular transfer phospholipids, cholesterol, gangliosides Processing plasma lipoproteins Milk-fat globule component
76677038 86821535 72537224 74354746 76614774 1620375
Journal of Proteome Research • Vol. 7, No. 4, 2008
32.64/9.01 34.37/9.58 13.12/10.88 58.54/8.3 60.79/9.26 148.97/7.97
2 5 1 3 5 27
research articles
Microsomal Proteome plasmic or cytoskeletal proteins, or the localization of newly synthesized proteins in transit from the ER. It is likely, however, that many of these proteins sediment with microsomes as artifacts possibly due to entrapment in membrane vesicles produced during homogenization or to adventitious binding interactions within homogenates. One of the benefits of proteomic analysis is that it is now possible to detect the extent to which membrane fractions contain extra-membrane components thus substantially supplementing information about protein associations and contamination previously made using a small number of marker enzymes.16 Proteins Involved in Lipid Metabolism, Transport, and Secretion. Triacylglycerols and other milk lipids are synthesized in the ER of mammary epithelial cells and are transported through the cytoplasm as milk secretory granules. The milk secretory granules fuse with the plasma membrane giving rise to secreted milk fat globules in a process controlled by the protein butyrophilin.17 We identified over 50 proteins involved in lipid metabolism, transport, and secretion including butyrophilin (Table 1). Some of these proteins, including acetylCoA carboxylase and fatty acid synthase, are cytoplasmic enzymes for fatty acid biosynthesis that can be detected by 2-DE of mammary homogenates. The microsomal pellet also contained cytoplasmic 6-phosphofructokinase and ATP-citrate lyase (citrate cleavage enzyme) that are required for fatty acid synthesis from glucose but were not detected by 2-DE of mammary homogenates.5 The presence of these enzymes in the washed microsomal pellet may indicate a functional association with ER or other organelles, although presence as artifactual contaminants of homogenization and subcellular fractionation is also likely. For example, the transcription factor STAT5B is an example of a soluble-nuclear protein, implicated in the control of lactation18 that was present in the microsomal pellet. We detected ER membrane enzymes involved in the metabolism of fatty acids including stearoyl-CoA desaturase and fatty acid desaturase 2 that introduce double bonds into fatty acids,19 plus prostaglandin I2 (prostacyclin) synthase and arachidonate 15-lipoxygenase that synthesize regulatory metabolites. We detected a single fatty acyltransferase, lysophosphatidic acid acyltransferase, that is responsible for adding fatty acids to the sn-2 position of triacylglycerols.20 Other enzymes known to be important in lactogenesis included an acyl-CoA synthetase long-chain family member which converts fatty acids to their CoA derivatives prior to esterification of glycerophosphate. It is possible that some enzymes for triacylglycerol synthesis were either too low abundance to detect or relatively resistant to digestion during the in-gel hydrolysis step. Other membrane proteins involved in lipid metabolism included receptor subunits and P450s with roles in steroid metabolism. Further fractionation of the microsomal pellet including by gradient centrifugation to separate ER derived vesicles and/or partition using Triton X-11421 may be needed before other ER integral membrane proteins can be detected. We detected dehydrogenase and hydratase subunits of the β-oxidation enzymes of mitochondria and peroxisomes.22 Vesicles derived from lysosomes, mitochondria, and peroxisomes are usual components of microsomal preparations.16 Also present were apolipoproteins, lipoprotein lipase, and low-density lipoprotein receptor-related protein subunits that are involved in delivery of lipids to mammary gland. Some other proteins we detected including retinol dehydrogenase and serine palmitoyltrans-
ferase do not appear to have been previously reported from normal mammary tissue. Our work complements protein databases established by proteomic analysis of a human mammary epithelial cell line23 and bovine milk fat globule membranes.24 It is notable that some of the enzymes we detected are known to be differentially expressedinbreastcancertissue,includingacetyl-CoAcarboxylase,25,26 fatty acid synthase,26 and 15-lipoxygenase,27 and that some of these proteins are suggested novel therapeutic targets.28 The mammary proteome that we have established, therefore, provides a basis for future investigation of specific mammary functions.
Conclusions We have carried out a proteomic survey of a microsomal subcellular fraction from lactating bovine mammary gland. Of the 703 proteins identified by 1-DE, 160 had predicted transmembrane domains. Fifty-two proteins were associated with lipid metabolism, transport, and secretion. The database that has been established develops a proteomic view of the metabolic potential of mammary gland. This can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation1 and to further dissection of normal and pathological processes in mammary tissue. Abbreviations: ACN, acetonitrile; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; ER, endoplasmic reticulum.
Acknowledgment. Support was from the New Zealand Foundation for Research Science and Technology (Grant VLAC 0301) and Fonterra (New Zealand) Ltd. Note Added after ASAP Publication. Figure 3 was incorrect on the version published ASAP on 3/7/2008. The correct version was published 3/12/2008. Supporting Information Available: Supplementary Tables S1–2 listing the proteins identified by 1-DE and RPLCESI-MS/MS and the homologies of unknown and hypothetical proteins in the microsomal fraction from the lactating bovine mammary tissues identified by 1-DE RPLC-ESI-MS/MS are available. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Rudolph, M. C.; McManaman, J. L.; Phang, T.; Russell, T.; et al. Physiol. Genomics 2007, 28, 323–336. (2) Aksu, S.; Scheler, C.; Focks, N.; Leenders, F.; et al. Proteomics 2002, 2, 1452–1463. (3) Jacobs, J. M.; Mottaz, H. M.; Yu, L. R.; Anderson, D. J.; et al. J. Proteome Res. 2004, 3, 68–75. (4) Davies, C. R.; Morris, J. S.; Griffiths, M. R.; Page, M. J.; et al. Proteomics 2006, 6, 5694–5704. (5) Beddek, A. J.; Rawson P.; Peng L.; Snell, R.; et al. Proteomics 2008, in press. (6) Wu, C. C.; Howell, K. E.; Neville, M. C.; Yates, J. R.; McManaman, J. L. Electrophoresis 2000, 21, 3470–3482. (7) Wu, C. C.; Yates, J. R.; Neville, M. C.; Howell, K. E. Traffic 2000, 1, 769–782. (8) Hoving, S.; Gerrits, B.; Voshol, H.; Muller, D.; et al. Proteomics 2002, 2, 127–134. (9) Hughes, S. M.; Moroni-Rawson, P.; Jolly, R. D.; Jordan, T. W. Electrophoresis 2001, 22, 1785–1794. (10) Peng, J.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. J. Proteome Res. 2003, 2, 43–50. (11) Chen, Y.; Kwon, S. W.; Kim, S. H.; Zhao, Y. J. Proteome Res. 2005, 4, 998–1005.
Journal of Proteome Research • Vol. 7, No. 4, 2008 1431
research articles (12) Bae, T. J.; Kim, M. S.; Kim, J. W.; Kim, B. W.; et al. Proteomics 2004, 4, 3536–3548. (13) Elias, J. E.; Haas, W.; Faherty, B. K.; Gygi, S. P. Nat. Methods 2005, 2, 667–675. (14) Luche, S.; Santoni, V.; Rabilloud, T. Proteomics 2003, 3, 249–253. (15) Balgley, B. M.; Laudeman, T.; Yang, L.; Song, T.; Lee, C. S. Mol. Cell. Proteomics 2007, 6, 1599–1608. (16) Lim, W. C.; Jordan, T. W. Biochem. J. 1981, 197, 611–618. (17) Robenek, H.; Hofnagel, O.; Buers, I.; Lorkowsi, S.; et al. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 10385–10390. (18) Wheeler, T. T.; Broadhurst, M. K.; Sadowski, H. B.; Farr, V. C.; Prosser, C. G Mol. Cell. Endocrinol. 2001, 176, 39–48. (19) Ntambi, J. M.; Miyazaki, M. Prog. Lipid Res. 2004, 43, 91–104. (20) Mistry, D. H.; Medrano, J. F. J. Dairy Sci. 2002, 85, 28–35. (21) Gilchrist, A.; Au, C. E.; Hiding, J.; Bell, A. W.; et al. Cell 2006, 127, 1265–1281.
1432
Journal of Proteome Research • Vol. 7, No. 4, 2008
Peng et al. (22) Bhaumik, P.; Koski, M. K.; Glumoff, T.; Hiltunen, J. K.; Wierenga, R. K. Curr. Opin. Struct. Biol. 2005, 15, 621–628. (23) Jacobs, J. M.; Mottaz, H. M.; Yu, L.-R.; Anderson, D. J.; et al. J. Proteome Res. 2004, 3, 68–75. (24) Reinhardt, T. A.; Lippolis, J. D. J. Dairy Res. 2006, 73, 406–416. (25) Chajès, V.; Cambot, M.; Moreau, K.; Lenoir, G. M.; Joulin, V. Cancer Res. 2006, 15, 5287–5294. (26) Migraum, L. Z.; Witters, L. A.; Pasternack, G. R.; Kuhajda, F. P. Clin. Cancer Res. 1997, 3, 2115–2120. (27) Jiang, W. G.; Watkins, G.; Douglas-Jones, A.; Mansel, R. E. Prostaglandins, Leukotrienes Essent. Fatty Acids 2007, 77, 67–77. (28) Lupu, R.; Mendendez, J. A. Endocrinology 2006, 147, 4056–4066.
PR700819B
