Dynamic and Differential in Vivo Modifications of the Isoform HMGA1a and HMGA1b Chromatin Proteins
2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês
10.1074/jbc.m407348200
ISSN1083-351X
AutoresDale D. Edberg, Joshua Adkins, David L. Springer, Raymond Reeves,
Tópico(s)Ubiquitin and proteasome pathways
ResumoMost naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types. Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types. Overexpression of the HMGA1 gene (formerly known as HMGIY (1Bustin M. Trends Biochem. Sci. 2001; 26: 152-153Abstract Full Text Full Text PDF PubMed Google Scholar)) is such a consistent feature of tumors that it has been suggested to be a "diagnostic" biochemical marker of both neoplastic transformation and cancer progression (2Bussemakers M.J. van de Ven W.J. Debruyne F.M. Shalken J.A. Cancer Res. 1991; 51: 606-611PubMed Google Scholar, 3Tamimi Y. van der Poel H.G. Karthaus H.F. Debruyne F.M. Shalken J.A. Br. J. Cancer. 1996; 74: 573-578Crossref PubMed Scopus (69) Google Scholar, 4Tallini G. Dal Cin P. Adv. Anat. Pathol. 1999; 6: 237-246Crossref PubMed Scopus (119) Google Scholar, 5Jansen E. Petit M.R. Schoenmakers E.F. Ayoubi T.A. van de Ven W.J. Gene Ther. Mol. Biol. 1999; 3: 387-395Google Scholar, 6Reeves R. Environ. Health Perspect. 2000; 108: 803-809Crossref PubMed Google Scholar, 7Galande S. Curr. Cancer Drug Targets. 2002; 2: 157-190Crossref PubMed Scopus (40) Google Scholar, 8Abe N. Watanabe T. Izumisato Y. Suzuki Y. Masaki T. Mori T. Sugiyama M. Fusco A. Atomi Y. J. Gastroenterol. 2003; 38: 1144-1149Crossref PubMed Scopus (25) Google Scholar). Elevated levels of HMGA1 gene products and HMGA1 proteins have been observed in almost every cancer type investigated and are correlated with increasing degrees of malignancy and metastatic potential of a number of cancer types (6Reeves R. Environ. Health Perspect. 2000; 108: 803-809Crossref PubMed Google Scholar, 9Reeves R. Beckerbauer L. Biochim. Biophys. Acta. 2001; 1519: 13-29Crossref PubMed Scopus (321) Google Scholar, 10Reeves R. Beckerbauer L.M. Prog. Cell Cycle Res. 2003; 5: 279-286PubMed Google Scholar). Additionally, it was demonstrated that overexpression of HMGA1 proteins in "normal" rat 1a cells and a human breast adenocarcinoma cell line caused neoplastic transformation and malignant metastatic progression of these cells, respectively (11Wood L.J. Maher J.F. Bunton T.E. Resar L.M. Cancer Res. 2000; 60: 4256-4261PubMed Google Scholar, 12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). Together, these results demonstrate that overexpression of full-length HMGA1 proteins in tumor cells is extremely widespread and biologically important. The HMGA1a (formerly known as HMG-I) and HMGA1b (formerly known as HMG-Y) isoform proteins are architectural transcription factors that are derived from alternatively spliced mRNA transcripts coded for by the HMGA1 gene located on human chromosome 6 (locus 6p21) with HMGA1b (95 amino acids; ∼10.6 kDa) containing an internal 11-amino acid deletion compared with HMGA1a (106 amino acids; ∼11.5 kDa) (13Friedmann M. Holth L.T. Zoghbi H.Y. Reeves R. Nucleic Acids Res. 1993; 21: 4259-4267Crossref PubMed Scopus (185) Google Scholar). Both isoforms contain three independent DNA-binding regions, called AT-hook motifs, that bind to the DNA minor groove and preferentially recognize the structure of short stretches of an AT-rich sequence (14Elton T.S. Nissen M.S. Reeves R. Biochem. Biophys. Res. Commun. 1987; 143: 260-265Crossref PubMed Scopus (39) Google Scholar, 15Reeves R. Nissen M. J. Biol. Chem. 1990; 265: 8573-8582Abstract Full Text PDF PubMed Google Scholar, 16Huth J. Bewley C.A. Nissen M.S. Evans J.N.S. Reeves R. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 657-665Crossref PubMed Scopus (307) Google Scholar). However, the internal 11-amino acid deletion changes the spacing between AT-hooks I and II in the HMGA1b protein compared with that in the HMGA1a isoform. Originally, the HMGA1a and -A1b proteins were thought to possess the same biological activities/functions because the binding of recombinant proteins (which lack secondary biochemical modifications) to DNA substrates appeared to be similar for both isoforms. The first suggestion that HMGA1a and -A1b proteins might have different biological functions in vivo came from the work of Banks et al. (17Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar) that demonstrated that post-translational modifications (PTMs) 1The abbreviations used are: PTMs, post-translational modifications; CIP, calf intestinal phosphatase; ESI, electrospray ionization; HA7C, transgenic HA-HMGA1a overexpressing MCF-7/Tet-Off cell line; MALDI MS, matrix-assisted laser desorption ionization mass spectrometry; MS/MS, tandem mass spectrometry/mass spectrometry; rh, recombinant human; RP-HPLC, reverse phase high performance liquid chromatography; TOF, time-of-flight. of the HMGA1a and HMGA1b isoforms affect their mode of DNA-protein interactions. Additionally, different PTM patterns were detected in vivo on HMGA1a and -A1b proteins isolated from different tissue culture cell types (17Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar). Direct confirmation that HMGA1a and HMGA1b could indeed have different functions in vivo was subsequently obtained when one or the other of these isoforms was overexpressed as a tetracycline-regulated transgene in human MCF-7 mammary epithelial tumor cells (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). These experiments demonstrated that artificially induced overexpression of the HMGA1b transgene in the non-metastatic MCF-7 cells caused them to progress much more rapidly to a metastatic and highly malignant phenotype than did induced overexpression of the transgenic HMGA1a isoform. Moreover, oligonucleotide microarray studies revealed that the gene expression profile in MCF-7 cells overexpressing transgenic HMGA1a was significantly different from the profile in cells overexpressing transgenic HMGA1b. Western blot analysis of proteins being expressed in these cells further confirmed the microarray results, thus demonstrating that the HMGA1a and HMGA1b isoforms differentially regulate specific genes in the transgenic MCF-7 mammary epithelial cells (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). Clearly, there are distinct functional differences between the HMGA1a and -A1b isoforms in vivo, but more research is required to determine whether PTMs of the proteins play a role in these observed biological differences. In vivo phosphorylation of HMGA1 was first discovered in Ehrlich ascites cells (18Lund T. Holtlund J. Laland S.G. FEBS Lett. 1985; 180: 275-279Crossref PubMed Scopus (40) Google Scholar) and Friend erythroleukemic cells (19Elton T.S. Reeves R. Anal. Biochem. 1986; 157: 53-62Crossref PubMed Scopus (73) Google Scholar) where, along with histone H1, they are among the most highly phosphorylated chromatin proteins in the nucleus. Further research also demonstrated that HMGA1 proteins are phosphorylated in a cell cycle-dependent manner, specifically during metaphase (20Lund T. Skalhegg B.S. Holtlund J. Blomhoff H.K. Laland S.G. Eur. J. Biochem. 1987; 166: 21-26Crossref PubMed Scopus (20) Google Scholar, 21Lund T. Laland S.G. Biochem. Biophys. Res. Commun. 1990; 171: 342-347Crossref PubMed Scopus (23) Google Scholar). These discoveries led researchers to identify HMGA1 phosphorylation by specific kinases such as cyclin-dependent kinase 2 (22Reeves R. Langan T.A. Nissen M.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1671-1675Crossref PubMed Scopus (115) Google Scholar, 23Meijer L. Ostvold A.C. Walass S.I. Lund T. Laland S.G. Eur. J. Biochem. 1991; 196: 557-567Crossref PubMed Scopus (43) Google Scholar, 24Nissen M.S. Langan T.A. Reeves R. J. Biol. Chem. 1991; 266: 19945-19952Abstract Full Text PDF PubMed Google Scholar), casein kinase II (25Palvimo J. Linnala-Kankkunen A. FEBS Lett. 1989; 257: 101-104Crossref PubMed Scopus (49) Google Scholar), protein kinase C (17Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar, 26Xiao D.M. Pak J.H. Wang X. Sato T. Huang F.L. Chen H.C. Huang K.P. J. Neurochem. 2000; 74: 392-399Crossref PubMed Scopus (51) Google Scholar), and homeodomain-interacting protein kinase-2 (27Pierantoni G.M. Fedele M. Pentimalli F. Benvenuto G. Pero R. Viglietto G. Santoro M. Chiariotti L. Fusco A. Oncogene. 2001; 20: 6132-6141Crossref PubMed Scopus (81) Google Scholar). In addition, other PTMs of HMGA1, such as acetylation by cAMP-response element-binding protein binding protein and p300/CBP-associated factors (28Munshi N. Merika M. Yie J. Senger K. Chen G. Thanos D. Mol. Cell. 1998; 2: 457-467Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 29Munshi N. Agalioti T. Lomvardas S. Merika M. Chen G. Thanos D. Science. 2001; 293: 1133-1136Crossref PubMed Scopus (182) Google Scholar) and protein methylation (17Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar, 30Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar, 31Sgarra R. Diana F. Rustighi A. Manfioletti G. Giancotti V. Cell Death Differ. 2003; 10: 386-389Crossref PubMed Scopus (24) Google Scholar), have been discovered. More recently, di-methylations of both arginine and lysine residues were discovered on the HMGA1a protein that may correlate to the metastatic potential of the cells (32Edberg D.D. Bruce J.E. Siems W.F. Reeves R. Biochemistry. 2004; 43: 11500-11515Crossref PubMed Scopus (46) Google Scholar). It has also been demonstrated that HMGA1 proteins are biochemically modified in vivo for specific purposes, for example, enhanceosome formation/destabilization (28Munshi N. Merika M. Yie J. Senger K. Chen G. Thanos D. Mol. Cell. 1998; 2: 457-467Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 29Munshi N. Agalioti T. Lomvardas S. Merika M. Chen G. Thanos D. Science. 2001; 293: 1133-1136Crossref PubMed Scopus (182) Google Scholar) and participation in apoptosis (30Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar, 31Sgarra R. Diana F. Rustighi A. Manfioletti G. Giancotti V. Cell Death Differ. 2003; 10: 386-389Crossref PubMed Scopus (24) Google Scholar, 33Diana F. Sgarra R. Manfioletti G. Rustighi A. Poletto D. Sciortino M.T. Mastino A. Giancotti V. J. Biol. Chem. 2001; 276: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Nevertheless, the present knowledge of the types and patterns of in vivo PTMs found on HMGA1 proteins in different cell types and in the same cells under different physiological conditions is quite limited, and must be better characterized before the biological significance of such modifications can be fully and critically assessed. An important unanswered question in cell biology is whether differences in the types and patterns of in vivo PTMs present on closely related isoform proteins differentially influence the biological function/activity of these proteins in cells. For example, a complete characterization of the types and sites of PTMs present on the HMGA1a and -A1b proteins found in the same cell type has not yet been conducted. Likewise, no systematic assessment has been made of the types and sites of PTMs found on these closely related isoform proteins in cells that have been experimentally induced to exhibit markedly different phenotypic characteristics to gain insight into the molecular modifications of the proteins relating to cellular phenotype. In the present study, we employed mass spectrometry techniques to examine systematically the in vivo PTMs found on the endogenous HMGA1a and -A1b proteins in cells under varying sets of conditions. We focused on the differential in vivo PTMs found on the closely related HMGA1a and HMGA1b proteins isolated from two genetically matched MCF-7 lines of human mammary epithelial cells ("MCF-7/Tet-Off" and HA7C) whose origins and biochemical and phenotypic characteristics have been described in detail previously (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). The parental MCF-7/Tet-Off cell line expresses very low levels of the endogenous HMGA1 protein, does not readily grow in soft agar, and does not form tumors in nude mice, which is characteristic of normal mammary epithelial cells (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). In contrast, the HA7C cell line (which was originally derived from the MCF-7/Tet-Off cell line by stable transfection) is induced to overexpress transgenic HMGA1a protein when the cells are grown in the absence of tetracycline. As a consequence of this induced overexpression of transgenic HMGA1a protein (∼40 times the amount of the endogenous protein and well within the concentration range found in naturally occurring human cancers), the HA7C cells acquire the ability to grow in soft agarose, form primary tumors when injected into nude mice, and exhibit a moderately metastatic and invasive phenotype (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). In contrast to previous studies that identified PTMs on only the most abundant HMGA1 protein species present in cells, an important technical advance in the present investigation was the inclusion of all of the different in vivo modified forms of the endogenous HMGA1 proteins found in a given cell type for analysis by mass spectrometry. Our results demonstrate that the endogenous HMGA1 proteins within a given cell type represent a complex and heterogeneous population, with the HMGA1a and HMGA1b proteins exhibiting a much higher level of in vivo PTMs than has been reported previously. Most importantly, we report the first observed differential in vivo modifications on the HMGA1a and HMGA1b proteins isolated from the same cell type, and we demonstrate that the PTM patterns found on these isoforms differ in non-metastatic and metastatic MCF-7 cells. Furthermore, complex patterns of PTMs were observed on all three of the AT-hook regions of the HMGA1a and -A1b proteins that appeared to differ between the isoforms present within the same cell type as well as between non-metastatic and metastatic cells. These findings provide the first comprehensive comparison of the PTM patterns found on the HMGA1 isoforms in mammalian cells. Cell Lines and Cell Culture Methods—The cell lines utilized in this study were MCF-7/Tet-Off (Clontech, catalog number C30071), which is a non-metastatic line of MCF-7 human breast adenocarcinoma cells, and HA7C, a derivative line of MCF-7/Tet-Off cells that has been stably transfected with an expression vector containing a full-length human HMGA1a cDNA (clone 7C) (34Johnson K.R. Lehn D.A. Reeves R. Mol. Cell. Biol. 1989; 9: 2114-2123Crossref PubMed Scopus (224) Google Scholar) in which transcription is controlled by a tetracycline-regulated promoter (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). In HA7C cells, the N terminus of the HMGA1a cDNA protein-coding region is fused, in-frame, with a hemagglutinin peptide tag so that the chimeric transgenic protein produced by the vector can be distinguished and separated from the native, endogenous HMGA1a protein. Both the parental MCF-7/Tet-Off and the HA7C cell lines were grown and maintained as described previously (12Reeves R. Edberg D.D. Li Y. Mol. Cell. Biol. 2001; 21: 575-594Crossref PubMed Scopus (219) Google Scholar). Tetracycline was not added to the medium of the HA7C cells in order to maximize the transgenic HMGA1a protein overexpression. The cells were harvested when confluent and frozen at –70 °C until needed. Protein Isolation, Purification, and Detection—Recombinant human (rh) HMGA1 proteins were extracted from Escherichia coli BL21 DE3 pLysS cells (Stratagene, La Jolla, CA) with diluted (5%) trichloroacetic acid and were purified by reverse phase high performance liquid chromatography (RP-HPLC) techniques, as described previously (35Reeves R. Nissen M.S. Methods Enzymol. 1999; 304: 155-188Crossref PubMed Scopus (58) Google Scholar). Briefly, following acid extraction, the HMGA1 proteins were purified by utilizing an RP-HPLC C4 Microsorb analytical column with a linear acetonitrile, 0.2% trifluoroacetic acid gradient from 10 to 25% acetonitrile. Further purification of the HMGA1 proteins was accomplished with a C18 Dynamax analytical RP-HPLC column with a linear acetonitrile, 0.2% trifluoroacetic acid gradient from 5 to 23% acetonitrile. RP-HPLC isolated HMGA1 protein purity was assessed by SDS-PAGE following standard protocols (34Johnson K.R. Lehn D.A. Reeves R. Mol. Cell. Biol. 1989; 9: 2114-2123Crossref PubMed Scopus (224) Google Scholar) and verified by MALDI MS. Endogenous HMGA1 proteins were also isolated from both of the MCF-7 cell lines by employing these conditions. Detection of Endogenous HMGA1 Proteins Isolated from MCF-7 Cells—To confirm the presence of HMGA1 proteins extracted from both parental and the transgenic MCF-7 cell lines and from RP-HPLC-purified protein fractions, SDS-PAGE and Western blot analysis using polyclonal anti-HMGA1 were employed following published protocols (35Reeves R. Nissen M.S. Methods Enzymol. 1999; 304: 155-188Crossref PubMed Scopus (58) Google Scholar). Additionally, SDS-PAGE was used to assess the purity and concentration of (within the nanogram to microgram range) endogenous, in vivo modified HMGA1 proteins using known concentrations of pure rhHMGA1 proteins as reference standards (data not shown). Enzyme Digestions of the HMGA1 Proteins—Purified native HMGA1 proteins from each of the experimental cells lines were separated into two equal fractions of 0.5 mg and lyophilized. One of the samples was dephosphorylated with calf intestinal phosphatase (CIP) (Roche Applied Science), and the reactions (3 μg of in vivo modified HMGA1, 30 units of CIP) were carried out at 37 °C for 12 h in the reaction buffer supplied by the manufacturer. The dephosphorylated HMGA1 proteins were purified from the CIP by trichloroacetic acid precipitation (35Reeves R. Nissen M.S. Methods Enzymol. 1999; 304: 155-188Crossref PubMed Scopus (58) Google Scholar). Both the native and dephosphorylated samples were then individually lyophilized, reconstituted, and divided into two equal fractions. Sequencing grade proteinase Arg-C (Sigma) or sequencing grade trypsin (Promega, Madison, WI) (50:1 mass ratio of protein to enzyme) in 50 mm ammonium bicarbonate (pH 8.0) at 37 °C was used to digest the samples. Tryptic digestions were carried out for 4 h, while Arg-C reactions were digested for 10 h, with the length of the reactions empirically determined. Partial tryptic digestions were essential because complete cleavage of HMGA1 resulted in small peptide fragments, making them difficult to analyze by mass spectrometry. Trifluoroacetic acid was added to a final volume of 2% to terminate the reactions. The samples were lyophilized, and the peptides were redissolved in H2O. MALDI Mass Spectrometry—A PerSeptive Biosystems Voyager DE-RP MALDI time-of-flight (TOF) mass spectrometer (Framingham, MA) was used to analyze the HMGA1 digestions in the linear positive ion mode according to published protocols (17Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar, 36Chong B.E. Lubman D.M. Rosenspire A. Miller F. Rapid Commun. Mass Spectrom. 1998; 12: 1986-1993Crossref PubMed Google Scholar, 37Mann M. Hendrickson R.C. Pandey A. Annu. Rev. Biochem. 2001; 70: 437-473Crossref PubMed Scopus (934) Google Scholar). TOF was measured over 256 laser pulses and averaged into a single spectrum. Saturated matrix solution consisting of 3,5-dimethoxy-4-hydroxycinnamic acid, 50% acetonitrile, 0.2% trifluoroacetic acid was mixed with full-length rhHMGA1 and purified in in vivo modified HMGA1 proteins. The HMGA1 protein digestions were then mixed with α-cyano-4-hydroxycinnamic acid matrix solution and calibration mixture 3 (Cal Mix) and analyzed by MALDI-TOF MS. Both matrices were purchased from Sigma. To maximize peak detection, three different scans were carried out on every protein/peptide sample. MALDI-TOF MS Calibration—The MALDI-TOF mass spectrometer was internally calibrated using standards purchased from PE Biosystems (Foster City, CA). Briefly, the best peak definition for the range of analysis (400–5000 mass/charge (m/z)) needed for the rhHMGA1a peptide samples was achieved with the instrument settings of noise filter "1" and Gaussian smooth "23." Internal calibrations were performed using the 379.35 m/z α-cyano-4-hydroxycinnamic acid matrix dimer and the 5734.59 m/z bovine insulin peaks, and these conditions were chosen for the first "rough" internal calibration. An additional fine-tuned calibration of each spectrum was accomplished by calibrating on multiple unmodified HMGA1 peptides resulting from either tryptic or Arg-C digestions. Data Analysis—Data Explorer version 5.1 (PE Biosystems) was used to analyze m/z ratios for proteins and peptides. The analysis strategies were as follows. Prior to analysis, known peptide peaks resulting from autodigestion of trypsin (prospector.ucsf.edu/ucsfhtml4.0/misc/trypsin.htm) and potential human keratin contamination masses identified by the EXPASY FindPept tool (www.expasy.ch) were removed from each tryptic spectrum. Autodigestion and human keratins resulting from Arg-C enzymatic digestions were also accounted for by using the Find-Pept tool and removed from the corresponding spectra. The remaining peak masses were then copied into an Excel (Microsoft, Redmond, WA) spreadsheet from the Data Explorer program on both the vertical axis and horizontal axis to produce a two-dimensional matrix. Each cell not on the vertical and horizontal axis in the spreadsheet matrix contained a formula that calculated mass differences between adjacent spectrum peaks. Mass differences between the 21.5- and 22.5-Da mass range from adjacent peaks, corresponding to possible sodium adducts, were removed from further analysis. Peak masses remaining after removal of the possible contaminating human keratin peaks, trypsin/Arg-C peptide peaks, and sodium adducts were further analyzed with the EXPASY FindMod tool (www.expasy.ch), Protein Prospector MS-digest program (www.prospector.ucsf.edu), and manual mass calculations. The average mass values of 14.03, 42.08, and 79.98 were used for the analysis of potential PTMs, methylation, acetylation, and phosphorylation, respectively. Only peaks that corresponded to our data range criterion of less than ±150 ppm were further analyzed. Electrospray Ionization Tandem Mass Spectrometry—HMGA1 peptides from enzymatic digestions were separated by using an Agilent 1100 capillary LC system with a 40-cm capillary column (150 μm inner diameter × 360 μm outer diameter, Polymicro Technologies, Phoenix, AZ) packed with 5-μm C18 particles (PoroS 20R2, Applied Biosystems, Foster City, CA). Peptide elution was achieved at a flow rate of 1.8 μl/min using water, acetonitrile, 0.1% acetic acid, 0.01% trifluoroacetic acid with a linear gradient from 10 to 60% acetonitrile. The capillary column flow was infused directly into a Thermo-Finnigan LCQ Deca XP ion trap mass spectrometer. The mass spectrometer duty-cycle length was optimized to include a single full MS scan followed by three MS/MS scans on the three most intense parental masses (determined by Caliber® software in real time) from the single parent ion full scan. Dynamic mass exclusion windows of 3 min were used. MS/MS spectra for all samples were measured with an overall mass/charge window of 400–2000 m/z. Analysis of HMGA1 Peptides—Resulting tandem mass spectra were analyzed by SEQUEST® (Bioworks 2.0 ThermoFinnigan) (38Eng J.K. McCormack A.L. Yates J.R. J. Am. Soc. Mass Spec. 1994; 5: 976-989Crossref PubMed Scopus (5444) Google Scholar, 39Yates III, J.R. McCormack A.L. Eng J.K. Anal. Chem. 1996; 68: 534-540Crossref PubMed Google Scholar, 40Yates III, J.R. Carmack E. Hays L. Link A.J. Eng J.K. Methods Mol. Biol. 1999; 112: 553-569PubMed Google Scholar, 41Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2074) Google Scholar, 42Washburn M.P. Wolters D. Yates III, J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4089) Google Scholar), which compares experimental spectra with predicted idealized mass spectra generated from a data base of protein sequences. These idealized spectra are weighted largely with "b" and "y" fragment ions, i.e. fragmentation at the peptide bond from the N and C termini, respectively. The peptide mass tolerance was set to 1.0; the fragment ion tolerance was set to 0.0; and trypsin and Arg-C enzyme rules were applied during SEQUEST® analysis. Single and double dynamic modifications representing phosphorylation, methylation, and acetylation were used to analyze the MS/MS spectra against a data base containing primarily HMG proteins and other chromatin proteins. Extensive manual analysis of singly and doubly ionized spectra was conducted (43Kinter M. Sherman N.E. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley-Interscience, New York2000Crossref Google Scholar) to validate and complete the analysis begun with SEQUEST®. Diverse Post-translationally Modified HMGA1 Isoform Protein Populations within Human MCF-7 Mammary Epithelial Cells—As illustrated in Fig. 1, the endogenous HMGA1a and HMGA1b proteins were isolated from the MCF-7/Tet-Off cells and scanned with MALDI-TOF MS to examine their PTMs. The most prominent peaks in the in vivo HMGA1 comprehensive profiles generally correspond to the proteins with a single acetylation, most likely N-terminal acetylation. Furthermore, HMGA1a (11.54–12.46 kDa) and HMGA1b (10.59–11.36 kDa) PTMs are observed over large mass ranges (Fig. 1). The array of different masses observed in the MALDI-TOF MS spectra of these protein preparations indicates that both the HMGA1a and -A1b proteins exist in cells as a heterogeneous population of many different biochemically modified forms. Furthermore, their different mass ranges suggest that some HMGA1a proteins are more highly modified in vivo than the HMGA1b isoforms. In Vi
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