Multivariable Difference Gel Electrophoresis and Mass Spectrometry

Abstract

Multivariable DIGE/MS was used to investigate proteins altered in expression and/or post-translational modification in response to activation of transforming growth factor (TGF)-β receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-β over time (0, 8, 24, and 40 h), and proteins were resolved using medium range (pH 4–7) and narrow range (pH 5.3–6.5) isoelectric focusing combined with up to 2 mg of protein to allow inspection of lower abundance proteins. Triplicate samples were prepared independently and analyzed together across multiple DIGE gels using a pooled sample internal standard to quantify expression changes with statistical confidence. Unsupervised principle component analysis and hierarchical clustering of the individual DIGE proteome expression maps provided independent confirmation of distinct expression patterns from the individual experiments and demonstrated high reproducibility between replicate samples. Fifty-nine proteins (including some isoforms) that exhibited significant kinetic expression changes were identified using mass spectrometry and database interrogation and were mapped to existing biological networks involved in TGF-β signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-β signaling. Multivariable DIGE/MS was used to investigate proteins altered in expression and/or post-translational modification in response to activation of transforming growth factor (TGF)-β receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-β over time (0, 8, 24, and 40 h), and proteins were resolved using medium range (pH 4–7) and narrow range (pH 5.3–6.5) isoelectric focusing combined with up to 2 mg of protein to allow inspection of lower abundance proteins. Triplicate samples were prepared independently and analyzed together across multiple DIGE gels using a pooled sample internal standard to quantify expression changes with statistical confidence. Unsupervised principle component analysis and hierarchical clustering of the individual DIGE proteome expression maps provided independent confirmation of distinct expression patterns from the individual experiments and demonstrated high reproducibility between replicate samples. Fifty-nine proteins (including some isoforms) that exhibited significant kinetic expression changes were identified using mass spectrometry and database interrogation and were mapped to existing biological networks involved in TGF-β signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-β signaling. 2D 1The abbreviations used are: 2D, two-dimensional; TGF, transforming growth factor; PCA, principle component analysis; HC, hierarchical clustering; P-, phospho-; MAPK, mitogen-activated protein kinase; ANOVA, analysis of variance; MOWSE, Molecular Weight Search; PPARA, peroxisome proliferative-activated receptor-α; PCNA, proliferating cell nuclear antigen; EF-Tu, elongation factor Tu. gel-based approaches are often used to survey the proteome on a global scale, typically resolving thousands of intact proteins based on charge (using isoelectric focusing) and apparent molecular mass (using SDS-PAGE). Despite its popularity for differential display proteomics, 2D gel-based strategies have until recently lacked the ability to directly quantify abundance changes in the same fashion as in stable isotope strategies using liquid chromatography coupled with tandem mass spectrometry (1Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags..Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4362) Google Scholar, 2Mason D.E. Liebler D.C. Quantitative analysis of modified proteins by LC-MS/MS of peptides labeled with phenyl isocyanate..J. Proteome Res. 2003; 2: 265-272Crossref PubMed Scopus (67) Google Scholar, 3Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. 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This use of a pooled sample internal standard provides every resolved protein form under survey with a unique internal standard across a coordinated set of DIGE gels (Refs. 9Alban A. David S.O. Bjorkesten L. Andersson C. Sloge E. Lewis S. Currie I. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard..Proteomics. 2003; 3: 36-44Crossref PubMed Scopus (783) Google Scholar and 10Friedman D.B. Hill S. Keller J.W. Merchant N.B. Levy S.E. Coffey R.J. Caprioli R.M. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry..Proteomics. 2004; 4: 793-811Crossref PubMed Scopus (323) Google Scholar; for a review, see Ref. 11Lilley K.S. Friedman D.B. All about DIGE: quantification technology for differential-display 2D-gel proteomics..Expert Rev. 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W. of two-dimensional electrophoresis with PubMed Scopus Google Scholar). resolving intact 2D gels from or post-translational and significant mass spectrometry that a set of is in the modified such changes be in the approaches mass on the modified of this was to the of DIGE/MS to quantify changes in protein abundance and/or post-translational modification on a global from multiple experimental each with independent biological this used as a transforming growth of human mammary epithelial cells with a expression This is of biological of the HER2/Neu (ErbB2) is in of breast the Rev. Mol. Cell. 2001; 2: PubMed Scopus Google Scholar). TGF-β is a that but and TGF-β and on PubMed Scopus Google Scholar) and been to with of in mammary (ErbB2) from mammary W. J. growth factor mammary U. S. A. 2003; PubMed Scopus Google Scholar, M.E. D. of mammary overexpressing transforming growth factor Cell. 2003; PubMed Scopus Google Scholar, I. E. M.H. 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Despite and is the molecular or of between TGF-β and the of potential for to TGF-β were over time (0, 8, 24, and 40 in experiments that were analyzed by on over protein was in the range mg of protein and in some and were by using narrow range isoelectric focusing (pH 5.3–6.5) in with up to 2 mg of protein gel. component analysis and hierarchical clustering of the individual and DIGE expression maps provided independent confirmation of distinct expression patterns from each and demonstrated high reproducibility between the replicate samples. experimental was using independent for statistical confidence and to to variation. of were identified using mass spectrometry and database and identified proteins were mapped to existing biological networks and to the of the proteins identified in cells were and as described S. N. of in human breast epithelial cells transforming growth factor cell 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) in a mixture of modified medium and medium with human growth factor and MCF10A cells in were with medium with human a of 2 8, 24, and 40 prior to cells were with and with and 2 each and for and the protein in the was using the protein was as described N. transforming growth in human cancer 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). were used for all maspin and cathepsin and was using the was using the each of were to a to the for by of using and for maspin were analyzed in internal standard of Friedman et al. D.B. Hill S. Keller J.W. Merchant N.B. Levy S.E. Coffey R.J. Caprioli R.M. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry..Proteomics. 2004; 4: 793-811Crossref PubMed Scopus (323) Google Scholar) and et al. Hill S. 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A method for the quantitative of protein in in the of and PubMed Scopus Google Scholar) and in of 2 of each sample was and combined into a single to the pooled sample internal for each experiment individual the pooled standard of each individual sample was labeled with of Cy3 or the pooled sample was labeled with of A was used such that the three samples from were labeled all with Cy3 or to for that 2 mg of protein as with mg of from each individual samples with of and the pooled sample internal standard with of of Cy3, and were used standard for was for on in the which the were with the of for each of for on in the and samples containing of were combined and with a aliquot of the pooled standard which an equal of 2 was mixtures were up to of with as for 2 which or was and the are from the have that to protein for protein to mass spectrometry D.B. Hill S. Keller J.W. Merchant N.B. Levy S.E. Coffey R.J. Caprioli R.M. 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Protein expression of the of Proteomics. 4: Full Text Full Text PDF PubMed Scopus Google Scholar). each DIGE samples for each gel were into and for by isoelectric focusing using a to the for a of were and the proteins were into the by the in with for by in for an was on gels using fluorescence with to the the of protein was for by until using a labeled proteome maps each DIGE gel were by and using a image were and for analysis using the of for the was and the gels were in for 2 and in in the This of protein and protein as the molecular and of the the apparent molecular mass of proteins during were on the same as as to protein of was used for DIGE was used to the of each individual protein from a or sample on a gel relative to the Cy2 from the pooled sample internal standard to the same each the fluorescent from each protein individual samples and internal are by the and abundance are made directly to the internal standard from the to run analytical for each for each individual gel were analyzed using the which for the of protein patterns and of and quantitative abundance for each protein between gels of a coordinated using the unique of each protein from the pooled internal this multiple each with independent experimental were with statistical confidence and the that every be made a single 2D DIGE gel. was associated with each in abundance or post-translational modification using and analysis of that the of expression a to the of between significant changes were the confidence for proteins resolved in a but changes the were Unsupervised and was using the the individual and samples based on the of expression patterns from the proteins identified in I. of protein expression are by each in the and by each in the expression the of a analysis into two principle and which the samples based on the two of in the the of the are the confidence a clustering of the samples based on of expression patterns in the which are as in an expression using a abundance from to expression were using and of proteins identified by mass spectrometry and database interrogation existing networks and was using time and in a of were and into peptides with modified and peptides were to a using an the samples were on the with an equal of in with and tandem was on a mass mass were in laser were to internal using the peptides and tandem were in a fashion based on the mass for each laser on each of the in each sample peptides and mass maps and the associated were used to in the and for to significant using the were protein molecular or isoelectric with of with of and with in the of proteins from was in the of but have an on the statistical of the proteins in this Molecular Weight of of with independent protein and of gel with molecular and were for each protein are in and cells were with 2 for 8, 24, and 40 and of was by of which directly by the TGF-β changes in DIGE/MS analysis using a pooled sample internal standard on every gel this experimental every DIGE gel in a set two of the samples with Cy3 or Cy5) with an equal aliquot of the internal standard an and sample be on the same the quantitative for a resolved protein is between the Cy3 or and the Cy2 internal standard for this between the Cy3 and for each resolved protein and are to the from the this for a protein the abundance from all samples be with statistical confidence in a experiment all coordinated by the same sample internal A of the the individual samples were labeled and into the DIGE gels with the internal is in this have a protein that is the time and the relative abundance is for this protein each gel relative to the from the protein in the internal standard and the and are between the gels using the Cy2 for that for this is in the abundance relative to the internal standard that is across all are all together to the relative and reproducibility of the expression DIGE analysis protein that exhibited significant changes in abundance or post-translational modification that were or and of the changes the confidence is for over but changes the were were from a of the gels and to protein using mass spectrometry and database interrogation as described under the of which are in and in identified unique proteins to post-translational modification or of proteins were as with isoelectric but apparent molecular with a post-translational the expression patterns of were to in and in were from for analysis used medium range for the isoelectric focusing this and mg of protein into each with range A. C. G. A. B. R. W. of two-dimensional electrophoresis with PubMed Scopus Google Scholar). the and to to lower abundance proteins in the same samples using narrow range isoelectric focusing (pH 5.3–6.5) in a set with protein gel and identified in the narrow range DIGE analysis to that were identified by mass spectrometry and database interrogation and of the proteome maps and DIGE/MS from the two range experiments significant DIGE in the narrow range that were of abundance to be by DIGE and/or mass spectrometry in the experiment and or were resolved in the range and proteins identified in the cathepsin and were by mass spectrometry in the narrow range experiment to in DIGE between the two 24, and proteins in the range and resolved in the narrow range gels and were by and inspection of the DIGE but were by mass spectrometry in for the narrow range experiment changes and/or statistical that have been in the experiment to the of in the to the experimental samples for of and to biological of the protein changes to by statistical and of the proteins identified by the of a analysis to display the two principle that between the two of the range distinct expression patterns from the and demonstrated high reproducibility between the replicate samples A and in the the expression for the of proteins in for each range A and the identified in the the principle component of the with by the principle the were of the and for and the that the of in the experiment is the time from the the relative of the between the and are to a of proteins used for the analysis in each were in an analysis of the protein expression patterns each sample and based on of the expression patterns of the proteins with to the in the each expression in the expression is the same as each in the expression and for each of the individual proteins in the analysis into in the expression and a on the is directly to the in which the mass spectrometry and the DIGE and were for the to over by and the biological of the protein expression changes in as individual samples to in this individual changes a to the of the proteins in by to networks and and the unique proteins by the protein identified in the and TGF-β time mapped to a of as a with this were and the peroxisome proliferative-activated that in proteins that were independently identified in the DIGE experiments is to biological the of proteins to networks provides into potential TGF-β and that have based on the of proteins in I. 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