Quantitative Proteomic Analysis Using Isobaric Protein Tags Enables Rapid Comparison of Changes in Transcript and Protein Levels in Transformed Cells

Abstract

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRβ has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRβ leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene. Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRβ has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRβ leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene. The development of approaches for measurement of relative expression of proteins between two (or more) samples is an essential aspect of systems biology. A common technique for this type of proteomic study has been two-dimensional gel electrophoresis, where proteins are separated by isoelectric point and molecular weight, and spot patterns are compared by sophisticated computer algorithms. Proteins of interest are then identified by MS. However, gel-based approaches have drawbacks, including amount of material required, reproducibility, and limiting of sensitivity by protein loss. These are balanced against recent advances in protein staining that allow intragel comparison of protein quantity from two or three samples (1Tonge R. Shaw J. Middleton B. Rowlinson R. Rayner S. Young J. Pognan F. Hawkins E. Currie I. Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.Proteomics. 2001; 1: 377-396Google Scholar). An alternative approach to proteomic analysis is LC-MS (2Washburn M.P. Wolters D. Yates 3rd, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Google Scholar). This provides an increased sensitivity compared with gel-based approaches and can catalogue protein present in a sample. However, relative quantitation of proteins using LC-MS is challenging. Quantification by analyzing two samples in parallel and comparing their mass spectrometric profiles is not feasible. Isotopic labeling of peptides, however, does allow two samples to be analyzed in a single experiment. Isotope-coded affinity tagging using ICAT reagent technology indicates peptide source, with peak height giving relative quantity (3Gygi 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: 994-999Google Scholar). Comparing the ICAT reagent approach with two-dimensional gel electrophoresis, however, demonstrates that neither offers comprehensive coverage of a proteome (4Patton W.F. Schulenberg B. Steinberg T.H. Two-dimensional gel electrophoresis; better than a poke in the ICAT?.Curr. Opin. Biotechnol. 2002; 13: 321-328Google Scholar). This is true in part because many proteins (and therefore peptides) do not contain cysteine, the amino acid used for covalent attachment of the isotopomer in ICAT reagents. Thus, much information can be discarded in the form of non-labeled peptides, whereas two-dimensional gel electrophoresis excludes many large, hydrophobic, and basic proteins. Stable isotope labeling with amino acids in cell culture uses isotopes of essential amino acids (for example deuterated leucine) to label cells in culture (5Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell Proteomics. 2002; 1: 376-386Google Scholar). The samples are mixed, proteolytically digested, and run in LC-MS experiments. All leucine-containing peptides appear as “heavy” and “light” peaks, giving relative protein abundance. This elegant method can only be used on cultured cells; it is unsuitable for study of primary material. For ICAT reagent and stable isotope labeling with amino acids in cell culture technologies, the labeled peptides have different masses in an MS scan; this increases the complexity of the MS spectra and necessitates that MS/MS is therefore performed on the same peptide (the heavy and light labeled versions) twice, wasting analysis time. Novel labeling reagents can overcome some of the limitations described above. Isobaric tags for relative and absolute quantitation (iTRAQ) 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; PDGFRβ, platelet-derived growth factor receptor subunit β; C/EBP, CCAAT/enhancer-binding protein. 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; PDGFRβ, platelet-derived growth factor receptor subunit β; C/EBP, CCAAT/enhancer-binding protein. use reagents that enable up to four samples to be analyzed within the same experiment. The labels consist of a protein-reactive group that labels all free amines (i.e. will label at the N terminus of all peptides and also the side chain of internal lysine residues), a balance group and a reporter group (6Ross 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. Multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell Proteomics. 2004; 3: 1154-1169Google Scholar). The labels are isobaric, with a different distribution of isotopes between the reporter and balance groups. Hence, each labeled peptide appears at the same mass in an MS scan, but upon fragmentation in the mass spectrometer, the label dissociates and releases the reporter group as a singly charged ion of masses 114.1, 115.1, 116.1, or 117.1, respectively. Relative peak area indicates the contribution of each sample to total peptide present, providing a measure of relative abundance. The balance group is also lost and the remaining peptide fragments, which all have addition of the same mass (i.e. the protein reactive group) provide data from which to infer the peptide sequence. The t(5,12) translocation found in chronic myelomonocytic leukemia results in the expression of the leukemogenic tyrosine kinase TEL/PDGFRβ and activation of the PDGFRβ tyrosine kinase domain (7Golub T.R. Barker G.F. Lovett M. Gilliland D.G. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation.Cell. 1994; 77: 307-316Google Scholar). This stem cell disease has been modeled by expressing the TEL/PDGFRβ in a multipotent hematopoietic stem cell line, FDCP-Mix. The effects of oncogenic expression can be subtle yet lead to profound changes in cellular development. Perhaps unlike signaling for proliferation or apoptotic suppression, the appropriate tools for immediate analysis of potential effectors of altered development are not freely available. Herein, we report the validation and use of iTRAQ reagents on the FDCP-Mix TEL/PDGFRβ system as a paradigm for rapid, systematic definition of oncogenic processes using proteomics and the value of iTRAQ in permitting direct comparison of transcriptome data. FDCP-Mix cells were transduced with TEL/PDGFRβ using a murine stem cell retroviral vector as described previously (8Pierce A. Owen-Lynch P.J. Spooncer E. Dexter T.M. Whetton A.D. p210 Bcr-Abl expression in a primitive multipotent haematopoietic cell line models the development of chronic myeloid leukaemia.Oncogene. 1998; 17: 667-672Google Scholar). Cells were routinely cultured in Fishers medium with 20% (v/v) horse serum supplemented with 10 ng/ml IL-3 (R&D Systems, Minneapolis, MN). Conditions for inducing myeloid differentiation FDCP-Mix cells were induced to differentiate by two methods as described previously (9Heyworth C.M. Dexter T.M. Kan O. Whetton A.D. The role of hemopoietic growth factors in self-renewal and differentiation of IL-3-de pend ent multipotential stem cells.Growth Factors. 1990; 2: 197-211Google Scholar). Briefly cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with pre-selected fetal calf serum (20% (v/v)) and a combination of recombinant murine granulocyte macrophage-colony-stimulating factor (50 units/ml; Biogen IDEC, Zug, Switzerland), recombinant human macrophage-colony-stimulating factor (5 ng/ml; Amgen Biologicals, Thousand Oaks, CA) plus recombinant murine IL-3 (0.1 ng/ml; Calbiochem, Nottingham, UK). Cells were prepared with a Cytospin centrifuge and stained with May Grunwald-Giemsa, and differential morphology was scored for greater than 100 cells per slide. Western blotting was carried out with standard protocols using a monoclonal antibody to the kinase domain of PDGFRβ or anti-phosphotyrosine antibodies (BD PharMingen, Oxford, UK). The phosphoprotein content (serine, threonine, and tyrosine) was measured by separation of total cell lysates on 10%T SDS-polyacrylamide gels and staining with Pro-Q diamond stain (Molecular Probes, Leiden, The Netherlands) per the manufacturer’s instructions. An overview of the workflow is shown in Fig. 1. 1–2 × 107 cells were lysed in 250 μl of 0.5 m triethylammonium bicarbonate (Sigma-Aldrich, St. Louis, MO) + 0.05% (w/v) SDS on ice for 20 min with regular vortexing. Protein was centrifuged at >10,000 × g for 20 min at 4 °C, supernatant was removed, and protein quantified using the modified Bradford protein assay (Bio-Rad Laboratories). Protein (50 or 100 μg) in 20 μl of 0.5 m triethylammonium bicarbonate/0.05% SDS was reduced by addition of 2 μl of 50 mm tris-(2-carboxyethyl)phosphine and incubation at 60 °C for 1 h. Reduced cysteine residues were then blocked by addition of 1 μl of 200 mm methylmethanethiosulfate in isopropanol and incubation at room temperature for a further 10 min. Protein was then digested by addition of 10 μl of trypsin at 0.5 μg/μl and incubated at 37 °C overnight. To label the peptides with iTRAQ reagent (Applied Biosystems, Warrington, UK), one unit of label (defined as the amount required to label 100 μg of protein) was thawed and reconstituted in 70 μl of ethanol, with vortexing for 1 min. The reagent solution was added to the digest and incubated at room temperature for 1 h. Labeling reactions were then pooled before analysis. To remove excess, unbound iTRAQ reagent and to simplify the peptide mixture before reversed-phase LC-MS/MS, peptides were washed and fractionated off line using a strong cation exchange column (Applied Biosystems). In brief, the peptide mixture was diluted 10-fold in loading buffer (10 mm potassium phosphate in 25% (v/v) acetonitrile, pH 3.0), and the pH was checked to ensure it remained between 2.5 and 3.3. Sample mixture was slowly injected onto the strong cation exchange cartridge and was washed with a further 1 ml of loading buffer to remove salts, tris-(2-carboxyethyl)phosphine, and unincorporated iTRAQ reagent. Samples were eluted from the column using 500-μl volumes of elution buffer (10 mm potassium phosphate in 25% (v/v) acetonitrile) containing increasing concentration of KCl. Salt concentrations used were 50, 100, 150, 200, 250, 300, 350, and 500 mm. Each salt fraction was then concentrated and dried in a SpeedVac (Thermo Electron, Waltham, MA). Dried peptide fractions were resuspended in 250 μl of 2% (v/v) acetonitrile/0.1% (v/v) formic acid. For each analysis, 60 μl of the peptide sample was loaded onto a column using an The Netherlands) and separated a from (v/v) acetonitrile/0.1% (v/v) formic acid to (v/v) acetonitrile/0.1% (v/v) formic acid to a mass spectrometer (Applied Biosystems). was using an data in for each the two most charged peptides to a 10 in the MS with between and were for Each peptide was and then for were against a protein from the (Applied Biosystems). The for iTRAQ reagent labels at N-terminal internal and and the cysteine as plus one within were with an MS of an MS/MS of and a of pooled data from all LC-MS of for peptide and protein identification is described in relative quantification of protein levels and levels in FDCP-Mix cells and of the as by is of the as by is of the as by is protein protein protein protein phosphoprotein subunit 2 factor factor factor factor acid protein phosphate protein group A heavy chain of protein kinase oncogene protein protein protein protein protein protein protein protein complex containing proteins as by iTRAQ protein molecular factor cell pend ent protein of the as by is in a from FDCP-Mix and TEL/PDGFRβ FDCP-Mix samples was prepared using in samples and then using up CA) per the manufacturer’s instructions. analysis was using murine by the microarray UK). changes were from the where appropriate analysis was FDCP-Mix cells were transduced to TEL/PDGFRβ with cells transfected with and TEL/PDGFRβ have shown that TEL/PDGFRβ has effects on tyrosine not However, TEL/PDGFRβ has but effects on protein tyrosine and also total phosphoprotein level and cell can growth expressing TEL/PDGFRβ M. M.H. Barker G.F. T.R. Gilliland D.G. The growth factor beta receptor beta in chronic myelomonocytic leukemia is a protein that and PDGF beta pend ent signaling S. A. Scholar). This not in cells. FDCP-Mix cells differentiate to form cells cultured in the appropriate E. C.M. A. Dexter T.M. and differentiation of pend ent multipotent stem cells are by cells and serum Scholar). of TEL/PDGFRβ this development that myeloid development to cells in control FDCP-mix cells in cells. 20 in culture, cells were present that primitive myeloid cell or cell This on differentiation to potential differences in transfected and cells using and proteomic To do the proteomic method required A that been digested with trypsin was used to the of quantitation of the iTRAQ reagents. The digest was and each was labeled with reagent or These labeled were mixed at and and analyzed by A is shown in Fig. Relative quantitation of proteins by iTRAQ reagent technology was accurate and for proteins The protein in the standard mixture was not with peptides to allow of and were that peptides remained by analysis of ion masses derived for the MS analysis and comparison with of the proteins. All isobaric of the iTRAQ reagent tag labeled The potential value of the system was further on lysates from mammalian FDCP-Mix cells and FDCP-Mix cells transduced with the TEL/PDGFRβ leukemic oncogene. In microarray data was for comparison from the FDCP-Mix cells described above. The is in Fig. 1. All four labels were the use of internal and within the same LC-MS for this paradigm This the of quantitation to be by including a ratio of control sample and a ratio of the TEL/PDGFRβ FDCP-Mix cell as shown in Fig. 1. an internal for the comparison of and cells is The was run in Labeling with iTRAQ reagents was and and quantitation was with of and samples were mixed and and samples were mixed is that were as can be In within the an with an ratio of and the FDCP-Mix a of the of the for peptides and proteins was In a peptides from proteins were of the of identified proteins and differences between samples in a total of proteins and is that a an the ratio of changes and a of the spectra by iTRAQ peptides are shown in Fig. where peptides between or by TEL/PDGFRβ expression are of MS/MS a from that does not show an expression upon TEL/PDGFRβ expression and the iTRAQ reagent used for relative quantitation MS/MS a from a protein that a in expression with TEL/PDGFRβ and the iTRAQ reagent used for relative quantitation the levels of the and labels compared with control protein labeled with and MS/MS a peptide an internal lysine labeled with iTRAQ reagent via the acid in side from protein that an in expression with TEL/PDGFRβ and the iTRAQ reagent used for relative quantitation the increased levels of the and labels compared with control with and changes are seen in from and control FDCP-Mix the of this oncogene on protein tyrosine This of in the transcriptome to changes using of microarray data with a a total of show a with TEL/PDGFRβ and show The data derived comparison of All of the differences identified by iTRAQ reagents with were giving a of 100 in total as of the data The ratio of the expression between FDCP-Mix and for each was and compared with the and and control from iTRAQ reagent The data from 100 proteins These were cell and protein the iTRAQ all differences were in the protein changes is with to on mechanisms of a of proteins changes in the proteome but not in the These a protein and factor This of the value of the iTRAQ approach therefore that it will transcriptome analysis. proteins identified using iTRAQ were not within the These A and and protein This was using alternative protein and iTRAQ therefore offers sample analysis. In the changes at and protein level cell protein and the changes in this were to the changes in their The iTRAQ reagent and microarray data also in that most of the changes identified are subtle or iTRAQ reagent technology is a method for relative quantification of proteins from up to four has potential to the sensitivity and of mass spectrometric analysis of the Validation of the approach is using a protein mixture and cell lysates from a disease iTRAQ reagent labels all peptides at their N with free amines in lysine side all of the peptide is peptides to be quantified from each protein and increasing the of the data by this have that it is with standard proteins and to label four peptide mixtures to using LC-MS/MS, to the relative of the peptide from four The labeling for iTRAQ reagents is with are for sample or of iTRAQ reagent approach is that the peptides from all samples appear as one peak in MS increasing the total ion for that the and only one MS/MS per peptide. to proteins were identified in a analysis of a μg of protein in a 100 protein using the same mass spectrometer and for peptide D. Whetton and A. However, the number of identified proteins in is than that for this relative of sensitivity is protein identification protocols are to the ion with the number of in each MS for A the quantification on proteins is by a combination of (for of M. and/or the of which the mass spectrometer to (defined by mass and the the MS/MS can be that the for MS/MS are the two a in the MS than the of this on one of the FDCP-Mix samples increased the number of proteins identified by a further not only a fraction of the sample is used in a single using the number of from each sample. proteomic analysis or can be using iTRAQ reagents and multidimensional plus ion in iTRAQ experiments. proteins are in the presence of shown to be increased by TEL/PDGFRβ is an protein that has been in A. S. F. P. D. A in to in 2002; and that is a of the leukemogenic oncogene D. B. by the p210 2004; Scholar). In study TEL/PDGFRβ expression levels, whereas have shown that increased expression of F. M. The for the can oncogene-mediated 3: Scholar). by is in myeloid H. M. O. P. expression in human hematopoietic levels in the cells provide as to the for this expression is by factors as and the M. of the proteins and S. A. Scholar). These proteins and of or to to cells A.D. of granulocyte and 2002; A.D. and differentiation to proliferation or growth 2002; Scholar). levels are in and are at the level. Thus, the data we have derived allow further on the of differentiation in cells. These can and for from in iTRAQ reagent to allow relative quantitation of factor In which is also reduced in is a in D.J. chromosomal and expression of the murine A. in 1999; it has a role in hematopoietic stem cell and differentiation and also a role in the differentiation in have been in D. J. K. K. M. with in and of J. P.J. factor the in the of that receptor from the of hematopoietic Scholar). Perhaps is that has been shown to to the and via a B. A. M. A. M. A. A that is of a peptide to the in and processes the 2004; Scholar). have a of this data with a microarray data from the same cell line provides levels of between and protein level. All of the changes from the sample were also by of all a level of However, the iTRAQ reagent approach identified changes in proteins that are by in that is seen in the levels of A comparison of this type was previously because stable isotope or gel-based approaches to on proteins expression than the relative abundances of all proteins in a sample. In we have shown that iTRAQ protein labeling reagents can be to proteins in which expression is This has the of using samples in a single The iTRAQ reagent data relative expression levels in up to four of the iTRAQ reagent data with microarray data a of all changes in a of the microarray are by iTRAQ reagent analysis. However, iTRAQ reagent also changes not by the iTRAQ reagent technology has value as a method for relative quantification of proteins in and cell