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• W2162188676 abstract "Mammals that enter deep hibernation experience extreme reductions in body temperature and in metabolic, respiratory, and heart rates for several weeks at a time. Survival of these extremes likely entails a highly regulated network of tissue- and time-specific gene expression patterns that remain largely unknown. To date, studies to identify differentially-expressed genes have employed a candidate gene approach or in a few cases broader unbiased screens at the RNA level. Here we use a proteomic approach to compare and identify differentially expressed liver proteins from two seasonal stages in the golden-mantled ground squirrel (summer and entrance into torpor) using two-dimensional gels followed by MS/MS. Eighty-four two-dimensional gel spots were found that quantitatively alter with the hibernation season, 68 of which gave unambiguous identifications based on similarity to sequences in the available mammalian database. Based on what is known of these proteins from prior research, they are involved in a variety of cellular processes including protein turnover, detoxification, purine biosynthesis, gluconeogenesis, lipid metabolism and mobility, ketone body formation, cell structure, and redox balance. A number of the enzymes found to change seasonally are known to be either rate-limiting or first enzymes in a metabolic pathway, indicating key roles in metabolic control. Functional roles are proposed to explain the changes seen in protein levels and their potential influence on the phenotype of hibernation. Mammals that enter deep hibernation experience extreme reductions in body temperature and in metabolic, respiratory, and heart rates for several weeks at a time. Survival of these extremes likely entails a highly regulated network of tissue- and time-specific gene expression patterns that remain largely unknown. To date, studies to identify differentially-expressed genes have employed a candidate gene approach or in a few cases broader unbiased screens at the RNA level. Here we use a proteomic approach to compare and identify differentially expressed liver proteins from two seasonal stages in the golden-mantled ground squirrel (summer and entrance into torpor) using two-dimensional gels followed by MS/MS. Eighty-four two-dimensional gel spots were found that quantitatively alter with the hibernation season, 68 of which gave unambiguous identifications based on similarity to sequences in the available mammalian database. Based on what is known of these proteins from prior research, they are involved in a variety of cellular processes including protein turnover, detoxification, purine biosynthesis, gluconeogenesis, lipid metabolism and mobility, ketone body formation, cell structure, and redox balance. A number of the enzymes found to change seasonally are known to be either rate-limiting or first enzymes in a metabolic pathway, indicating key roles in metabolic control. Functional roles are proposed to explain the changes seen in protein levels and their potential influence on the phenotype of hibernation. Mammalian hibernators display the physiological traits of a nonhibernator or homeotherm in the summer months, but in the winter function in a heterothermic manner. They spend most of the winter in a state of deep torpor during which body temperature is as low as –2.9 °C (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), and there are concomitant extreme reductions in heart, respiratory, and metabolic rates (reviewed in Refs. 2Boyer B.B. Barnes B.M. Molecular and metabolic aspects of mammalian hibernation..BioScience. 1999; 49: 713-724Google Scholar and 3Carey H.V. Andrews M.T. Martin S.L. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature..Physiol. Rev. 2003; 83: 1153-1181Google Scholar). These “bouts” of torpor last from as few as 5 days to as many as 3Carey H.V. Andrews M.T. Martin S.L. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature..Physiol. Rev. 2003; 83: 1153-1181Google Scholar, 4Lyman C.P. Willis J.S. Malan A. Wang L.C.H. Hibernation and torpor in mammals and birds.in: Physiological Ecology. Academic Press, New York1982: 92-103Google Scholar, 5Boyer B.B. Barnes B.M. Lowell B.B. Grujic D. Differential regulation of uncoupling protein gene homologues in multiple tissues of hibernating ground squirrels..Am. J. Physiol. Regul. Integr. Comp. Physiol. 1998; 275: R1232-R1238Google Scholar weeks depending on the species. They are separated by arousals to euthermy that are 10–14 h on average for our model species, the golden-mantled ground squirrel, Spermophilus lateralis (unpublished calculations, see also Fig. 1). The arousals to euthermy are driven by endogenous mechanisms of rewarming and appear to be essential for survival, because the animals persist in regular arousals despite their high energetic expense (4Lyman C.P. Willis J.S. Malan A. Wang L.C.H. Hibernation and torpor in mammals and birds.in: Physiological Ecology. Academic Press, New York1982: 92-103Google Scholar).As in any organism, homeostasis is maintained by means of the fine-tuning of cellular processes that find their basis in differential patterns of gene expression and protein activity. Gene expression in the summer active liver is expected to resemble that of any euthermic mammalian liver; whereas during the hibernation season this expression pattern could change to reflect the unique biochemistry of hibernation. Such changes may adapt the animal for survival of heterothermy and other aspects of the torpid phenotype. Hibernation research has discovered examples of differential gene expression at the mRNA and protein levels (5Boyer B.B. Barnes B.M. Lowell B.B. Grujic D. Differential regulation of uncoupling protein gene homologues in multiple tissues of hibernating ground squirrels..Am. J. Physiol. Regul. Integr. Comp. Physiol. 1998; 275: R1232-R1238Google Scholar, 6Fahlman A. Storey J.M. Storey K.B. Gene up-regulation in heart during mammalian hibernation..Cryobiology. 2000; 40: 332-342Google Scholar, 7Gorham D.A. Bretscher A. Carey H.V. Hibernation induces expression of moesin in intestinal epithelial cells..Cryobiology. 1998; 37: 146-154Google Scholar, 8Kondo N. Kondo J. Identification of novel blood proteins specific for mammalian hibernation..J. Biol. Chem. 1992; 267: 473-478Google Scholar, 9O’Hara B.F. Watson F.L. Srere H.K. Kumar H. Wiler S.W. Welch S.K. Bitting L. Heller H.C. Kilduff T.S. Gene expression in the brain across the hibernation cycle..J. Neurosci. 1999; 19: 3781-3790Google Scholar, 10Epperson L.E. Martin S.L. Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation..Physiol. Genomics. 2002; 10: 93-102Google Scholar, 11Andrews M.T. Squire T.L. Bowen C.M. Rollins M.B. Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal..Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8392-8397Google Scholar, 12Srere H.K. Wang L.C. Martin S.L. Central role for differential gene expression in mammalian hibernation..Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7119-7123Google Scholar). All of these differentially expressed genes are present in the genomes of nonhibernators, and their basic function was elucidated in nonhibernating animals. In the hibernator, the temptation is to search for genes that are unique to hibernators and demonstrate special function at low temperatures, but to date none of these “super genes” have been identified. Instead, current data lend credence to the proposed concept that differential expression of mammalian genes provides the basis for the phenotype of hibernation, rather than newly derived hibernation-specific proteins and enzymes (12Srere H.K. Wang L.C. Martin S.L. Central role for differential gene expression in mammalian hibernation..Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7119-7123Google Scholar). This conservation among mammals is significant in that it suggests it will be possible to utilize new bioinformatics approaches despite the lack of genomic information for ground squirrel, and also that it provides basis for medical applications that derive from an understanding of hibernation (13van Breukelen F. Martin S.L. Molecular adaptations in mammalian hibernators: Unique adaptations or generalized responses?.J. Appl. Physiol. 2002; 92: 2640-2647Google Scholar).Research on differential gene expression in hibernating mammals has been primarily at the nucleic acid level in the identification of mRNAs whose expression varies seasonally. For more abundant RNAs, as most methods favor, there is a positive correlation between the steady-state levels of proteins and their corresponding mRNAs (14Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast..Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar). However, the RNA studies are limited in that they remain at least one step removed from the phenotype. But protein abundance is a step closer to functionality, and technology is advancing such that these studies are now becoming feasible. The application of two-dimensional (2D) 1The abbreviations used are: 2D, two-dimensional; SA, summer active; Ent, entrance; ET, early torpor; LT, late torpor; Ar, Arousing; IBA, interbout aroused; ID, identification; FABP, fatty acid-binding protein; FTHFD, 10 formyltetrahydrofolate dehydrogenase; 10 formyl THF, 10 formyltetrahydrofolate; PEPCK, phosphoenolpyruvate carboxykinase 2; PEP, phosphoenolpyruvate. 1The abbreviations used are: 2D, two-dimensional; SA, summer active; Ent, entrance; ET, early torpor; LT, late torpor; Ar, Arousing; IBA, interbout aroused; ID, identification; FABP, fatty acid-binding protein; FTHFD, 10 formyltetrahydrofolate dehydrogenase; 10 formyl THF, 10 formyltetrahydrofolate; PEPCK, phosphoenolpyruvate carboxykinase 2; PEP, phosphoenolpyruvate. SDS-PAGE with LC-MS/MS enables a broad spectrum, noncandidate approach to analysis of relative protein expression (“proteomics”). Following these established approaches with newly developed staining and scanning methods results in a powerful means to quantitatively assess several hundred steady-state protein levels in multiple stages of hibernation. However, the use of proteomics in cross-species analysis such as this is still in its infancy; and ground squirrels as rodents are only distantly related to rat and mouse (15Adkins R.M. Walton A.H. Honeycutt R.L. Higher-level systematics of rodents and divergence time estimates based on two congruent nuclear genes..Mol. Phylogenet. Evol. 2003; 26: 409-420Google Scholar), the closest model organisms. This study establishes the effectiveness of proteomic approaches with regard to ground squirrel sequences.The liver was chosen for this study due to its critical role in a number of processes that are likely to be crucial for survival of hibernation. Ground squirrels store fuel in the form of triacylglycerols in the adipose tissue and rely largely on this stored fat for survival of the winter (Ref. 16Wilson B.E. Deeb S. Florant G.L. Seasonal changes in hormone-sensitive and lipoprotein lipase mRNA concentrations in marmot white adipose tissue..Am. J. Physiol. 1992; 262: R177-R181Google Scholar and references therein). The processes of lipid metabolism include the lipolytic enzymes and proteins involved in transport of fatty acids, many of which are synthesized in the liver. The liver is also the site of synthesis for a number of enzymes involved in gluconeogenesis and ketone body formation, both processes required for fuel generation during the hibernation season (17Galster W.A. Morrison P.R. Gluconeogenesis in arctic ground squirrels between periods of hibernation..Am. J. Physiol. 1975; 228: 325-330Google Scholar, 18Gehnrich S.C. Aprille J.R. Hepatic gluconeogenesis and mitochondrial function during hibernation..Comp. Biochem. Physiol. B. 1988; 91: 11-16Google Scholar, 19Krilowicz B.L. Ketone body metabolism in a ground squirrel during hibernation and fasting..Am. J. Physiol. 1985; 249: R462-R470Google Scholar, 20LeBlanc P.J. Obbard M. Battersby B.J. Felskie A.K. Brown L. Wright P.A. Ballantyne J.S. Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus..J. Comp. Physiol. B. 2001; 171: 327-334Google Scholar). Another major function of hepatocytes is the synthesis of bile, which is used by the gut for emulsification of dietary fats. During fasting, the requirements for bile acids may be greatly reduced and the lack of bile would affect cholesterol metabolism and some aspects of lipid mobility. Cholesterol not used for bile acid synthesis might be shunted for use in hormone synthesis, e.g. for use in the adrenal synthesis of corticoids. In addition to liver-specific alterations in protein expression, a number of processes are expected to be affected in most cell types. For example, the cellular machinery required for protein protection, stability, and turnover and for redox balance in the cell is expected to undergo changes with respect to season (3Carey H.V. Andrews M.T. Martin S.L. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature..Physiol. Rev. 2003; 83: 1153-1181Google Scholar). These are a few of the many processes that could be affected at the protein level in the hibernator.The goal of this work was to identify liver proteins that are differentially expressed or modified during hibernation. Initially, 2D gel comparisons were made between three stages of the hibernation cycle: summer active (SA), interbout aroused (IBA), and late in torpor (LT) (see Fig. 1). Each group had a sample size of four, and a number of differences were found. However, only seven spots were found that changed with significance (p < 0.05), and SA levels were higher than IBA and/or LT in all of these (21Epperson L.E. Out cold: Liver gene expression in a mammalian hibernator, the golden-mantled ground squirrel.in: Molecular Biology Program. University of Colorado HSC, Denver, CO2003: 70-92Google Scholar). We noted a great deal of individual variability that precluded determination of many statistical differences without a larger sample size. This variability along with a consistent lack of equivalent resolution in the LT gels (22Martin S.L. Dahl T. Epperson L.E. Slow loss of protein integrity during torpor: a cause for arousal?.in: Life in the Cold: Evolution, Adaptation, Mechanisms and Applications. Institute of Arctic Biology, Fairbanks, AK2004: 119-208Google Scholar) led to an experimental redesign. Earlier we proposed that interbout arousals might be essential to restore gene products that are slowly lost during torpor (23Martin S.L. Srere H.K. Belke D. Wang L.C.H. Carey H.V. Differential gene expression in the liver during hibernation in ground squirrels.in: Life in the Cold. Westview Press, Boulder, CO1993: 443-453Google Scholar). If this is the case, then animals re-entering torpor after a replenishing period of interbout arousal will have fully restored their complement of required proteins; hence, protein samples from nine SA animals were compared with the same from nine entrance (Ent) animals. Ent animals are those that had been euthermic (35–37 °C body temperatures) for a complete arousal, ∼10–14 h, and they were sacrificed upon their descent into torpor (body temperatures range from 30 °C to 16 °C, see Fig. 1).Here we report the results from a comparison of total liver samples from two seasonal stages: SA and winter hibernation, specifically during entrance into torpor (Ent; Fig. 1). We found 84 reproducible 2D gel spots that changed in steady-state level seasonally with statistical significance (p < 0.05). Two-thirds of these were higher in entrance than in summer. Only six spots were not identified using the available databases, 10 others contained more than one protein, and 68 gave unique identifications. The power of this approach lies in the fact that hundreds of protein changes are assessed quantitatively in a single experiment, generating an unbiased and broad view of alterations in the biochemical pathways of hibernation. Collectively, the results from this study significantly enhance our current comprehensive understanding of the biochemical basis of the hibernating phenotype.EXPERIMENTAL PROCEDURESAnimals and Acquisition of Tissue—Golden-mantled ground squirrels (S. lateralis) to be sacrificed during hibernation were trapped in late August and abdominally surgically implanted with radio telemeters (VM-FH discs; Mini Mitter Co., Inc., Sunriver, OR) prior to the onset of hibernation for precise remote monitoring of body temperatures (10Epperson L.E. Martin S.L. Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation..Physiol. Genomics. 2002; 10: 93-102Google Scholar). After healing from surgery (∼2 weeks), the animals were moved to an environmental chamber. Each cage housed a single animal and was placed on top of a receiving pad to capture and transmit the radiotelemetry signal of body temperature. The temperature in the chamber was lowered stepwise to 5 °C over 2 weeks, where it was maintained for the hibernation season. To mimic burrow conditions, the animals were kept in constant darkness without food and water while the chamber was at 5 °C. Telemetry data were collected every 10 min on a computer in an adjacent room using Datacol 3 software. The SA animals were trapped in May-July, maintained at 22 °C in a 12-hour light-dark cycle with food and water ad libitum and sacrificed for tissue collection within 12–36 h. Animals were sacrificed according to protocol in either summer (SA), body temperature (Tb) approx. 37 °C or entrance (Ent), Tb ranging from 30 °C to 16 °C; CO2 asphyxiation was used for all animals. Livers were removed, snap frozen in N2 (l), and stored at −80 °C until needed. The animals used for this study were sacrificed on the following dates: SA animals: 30 May (2 animals), 3 June (2Boyer B.B. Barnes B.M. Molecular and metabolic aspects of mammalian hibernation..BioScience. 1999; 49: 713-724Google Scholar), 23 June (2Boyer B.B. Barnes B.M. Molecular and metabolic aspects of mammalian hibernation..BioScience. 1999; 49: 713-724Google Scholar), 25 July (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 27 July (2Boyer B.B. Barnes B.M. Molecular and metabolic aspects of mammalian hibernation..BioScience. 1999; 49: 713-724Google Scholar); Ent animals: 1 Dec (2Boyer B.B. Barnes B.M. Molecular and metabolic aspects of mammalian hibernation..BioScience. 1999; 49: 713-724Google Scholar), 18 Dec (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 27 Dec (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 12 Jan (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 17 Jan (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 18 Jan (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 19 Jan (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar), 26 Jan (1Barnes B.M. Freeze avoidance in a mammal: Body temperatures below 0 °C in an arctic hibernator..Science. 1989; 244: 1593-1595Google Scholar). All animal care and use procedures were approved by the University of Colorado Institutional Animal Care and Use Committee.Tissue Preparation—For each animal, ∼200 mg of frozen liver was removed from −80 °C and homogenized using a Polytron (Brinkmann Instruments, Westbury, NY) in a sucrose buffer containing protease inhibitors, i.e. 0.5 m sucrose, 0.1 m phosphate, 5 mm MgCl2, 1 mm PMSF, 10 μg/ml each of chymostatin, leupeptin, antipain, and pepstatin. Homogenate was passed through a 25-gauge needle 10 times and centrifuged 10 min at 4 °C, 500 × g. The supernatant was divided into small (20-μl) aliquots, snap frozen in liquid nitrogen, and stored again at −80 °C until use. One aliquot was removed to determine total protein content using a BCA Protein Assay Reagent Kit (Pierce, Rockford, IL).2D Gels—A methanol/chloroform precipitation was used to remove lipids from 200 μg of total liver protein at room temperature as described (24Wessel D. Flugge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids..Anal. Biochem. 1984; 138: 141-143Google Scholar). The entire precipitate from the 200 μg was solubilized in sample buffer first by repeat pipetting, then shaking in a Thermomixer R (Eppendorf, Westbury, NY) at 30 °C for 30–60 min at 900 rpm (25Taylor R.S. Wu C.C. Hays L.G. Eng J.K. Yates 3rd, J.R. Howell K.E. Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation..Electrophoresis. 2000; 21: 3441-3459Google Scholar). This protein solution was used to rehydrate a dried first-dimension strip (Immobiline DryStrip, pH 3–10 nonlinear, 18 cm; Amersham Pharmacia Biotech, Piscataway, NJ) overnight at room temperature under mineral oil. The strips were subjected to IEF (26Bernard K.R. Jonscher K.R. Resing K.A. Ahn N.G. Methods in functional proteomics: Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in-gel digestion and identification of proteins by mass spectrometry..Methods Mol. Biol. 2004; 250: 263-282Google Scholar) using a Multiphor II apparatus (Amersham Pharmacia Biotech) with focusing parameters as follows (in hh:mm): step to 500 V, 00:15; ramp to 3,500 V, 01:30; hold at 3,500 V, 15:00. After IEF, the first-dimension strips were reduced and alkylated in DTT and iodoacetamide, and the proteins were separated by size in the second dimension by SDS-PAGE on a 9–16% gradient gel. Note: each sample was run repeatedly (2–5×) until the same sample yielded two gels that were of identical high quality and useful for individual spot comparison across the whole gel. The gels were stained using SYPRO Ruby (Bio-Rad, Hercules, CA) as follows: all six gels from one set were placed together in 1 liter of fix for 1 h (10% methanol, 7% acetic acid), then overnight in 1 liter of SYPRO Ruby with gentle rocking in a foil-covered plastic container at room temperature. Stain was used repeatedly (5–6×) in separate experiments. The gels were removed into fresh fix solution and left to destain during the day; fix was replaced for another overnight destaining. The gels were scanned on a Typhoon 9400 (Molecular Dynamics, Sunnyvale, CA) using the green or blue laser for excitation. Each gel was scanned and the photomultiplier tube voltage adjusted until the most intense spots were in the linear range, just short of saturating, allowing for subsequent quantitation of all the spots on each gel. A control scanning experiment showed that photobleaching was negligible, as the same region scanned 10 times resulted in the same pixel volume each time, indicating that there was no loss of pixel signal with multiple scans. Gel images were analyzed with Melanie 4 software (GeneBio, Geneva, Switzerland). The group identification (ID), that is, the single number that represents the same protein spot on many gels, was assigned with the automated feature of Melanie 4 software followed by a significant amount of “manual” matching. The t test statistics were determined with the use of Excel software (Microsoft Corporation, Redmond, WA).In-gel Tryptic Digests—Gel spots were excised with clean razor blades on a UV light box and subsequently digested with trypsin essentially as described (25Taylor R.S. Wu C.C. Hays L.G. Eng J.K. Yates 3rd, J.R. Howell K.E. Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation..Electrophoresis. 2000; 21: 3441-3459Google Scholar); although extraction times were increased for greater peptide recovery to 3× for 1 h each.MS and Protein Identification—The dried tryptic fragments from a single spot were resuspended in a small volume (10 μl) of 5% formic acid. This sample was loaded using a pressure bomb onto a narrow-bore (0.150-mm inner diameter) fused silica column that contained C18 reversed-phase packing material (Aqua C18/ODS 5-μm particle size (Phenomenex, Inc., Torrance, CA) from cracked column p/n 00A-4299E0). The sample was eluted from the column into the mass spectrometer (LCQ-Deca; ThermoFinnigan, San Jose, CA) by HPLC (Agilent 1100 series pump; Agilent Technologies, Wilmington, DE) and nanospray using a hydrophobicity gradient over 30 min. The buffers that comprised the gradient were 5% ACN, 0.1% formic acid (buffer A) and 80% ACN, 0.1% formic acid (buffer B). Full and tandem mass spectra were collected for each spot and analyzed using XCalibur and Sequest software (ThermoFinnigan), followed by a stringent filter, DTASelect (27Tabb D.L. McDonald W.H. Yates 3rd, J.R. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics..J. Proteome Res. 2002; 1: 21-26Google Scholar). All of the positive matches are based on sequence identity at the peptide level and sequence homology at the protein level to sequences in the mammalian RefSeq database (downloaded December 2003: “vertebrate_mammalian,” ftp.ncbi.nih.gov/refseq/release), because it contains very few ground squirrel sequences. Additionally, many spectra were analyzed against a small golden-mantled ground squirrel database, which was generated by translation of a partial cDNA database available at legr.liv.ac.uk under squirrelBASE. Although most proteins identified were absent from this database, if it happened that a protein was present, the peptide coverage was generally higher than for the mammalian database.During the spectral collection, the full MS scan was followed by tandem mass spectral (MS/MS) scans of the three highest peaks, with a dynamic exclusion of 2 min. The parameters used for the initial Sequest search were as follows: parent and fragment masses were both set to monoisotopic, low to high mass limits for the precursor were 700–4,000 m/z (about 6 aa minimum), maximum number of internal cleavage sites was 2, peptide mass tolerance was 1.0, fragment ion tolerance was 0.0, trypsin was the enzyme used initially followed by a search with “no enzyme” if an ID was not obtained from the first run, searches were performed for covalently modified (alkylated) cysteines with a mass shift of +57. No statistics were applied. The DTASelect output is a list of proteins comprising virtually unambiguous IDs. Short peptides are filtered out, as are peptides with low cross-correlation scores from Sequest (XCorr) (27Tabb D.L. McDonald W.H. Yates 3rd, J.R. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics..J. Proteome Res. 2002; 1: 21-26Google Scholar). The thresholds for DTASelect were set as follows: minimum XCorr for +1, 1.8; +2, 2.5; +3, 3.5; minimum DeltaCN, 0.08; minimum peptides per locus, 2. This list, for a single spot, often includes human keratin, trypsin, and a single other ID, which is the actual protein ID. However, 6 spots gave no ID and 10 spots gave more than one ID. Conclusions regarding a change in abundance of any of the proteins comprising these 10 spots will require independent measurements.The accession numbers and species listed in Table I, Table II, Table III were selected from a typically multi-species list of protein matches (see supplemental table). They represent the best protein ID in that their peptide recovery resulted in either the highest protein coverage found or was equivalent to the high coverage found in the homologous protein from another species. The peptide number as given in Tables I and II were tabulated conservatively due to the cross-species nature of this study. Frequently, multiple peptides are recovered in the mass spectrometer, each with its own fragmentation spectrum, that are found to overlap one another on the protein sequence; often these peptides differ in length by a single amino acid. We have chosen not to count these as separate peptides; rather, if the sequence coverage for one peptide is completely “accounted for” by another recovered peptide, this was counted as a single peptide. However, if two peptides partially overlapped, they were counted separately. For example, for group ID 502, three of the peptides recovered were: N.TVIVKPAEQTPL.T, K.PAEQTPLTALHVASLIK.E, and L.TALHVASLIK.E. The first two overlap by several residues, but each covers unique sequence, so they were counted as two separate peptides. The third peptide was not counted because it is completely contained within the second peptide.Table IProtein identifications for spots whose entrance levels are greater than summerIDpFold changen, SAn, EntProtein identification(s)Abbrev.MWNo. of pept.AccessionSpecies1192.4186Pyruvate carboxylasePC129633811761615Human1201.8899Pyruvate carboxylasePC12963314632808Human121*2.0898Pyruvate carboxylase (probable ID)126*2.3879Saccharopine dehydrogenaseO95462102147113027640Human127*1.7799Saccharopine dehydrogenase (probable ID)1911.3499Valosin-containing protein (ER ATPase)TERA89321166005942Human2211.5199MitofilinIMMT83677934855983Rat2221.3098No ID obtained245*1.3599BiP (glucose-regulated protein 78)GR7872333516507237Human246*1.5299NADH coenzyme Q reductaseNUAM79573633519475Human2591.4199Dipeptidylpeptidase" @default.
• W2162188676 created "2016-06-24" @default.
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• W2162188676 creator A5019887423 @default.
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• W2162188676 date "2004-09-01" @default.
• W2162188676 modified "2023-10-15" @default.
• W2162188676 title "Quantitative Analysis of Liver Protein Expression During Hibernation in the Golden-mantled Ground Squirrel" @default.
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