Quantitative analysis of both protein expression and serine / threonine post‐translational modifications through stable isotope labeling with dithiothreitol

While phosphorylation and O‐GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post‐translational modifications have been difficult to analyze in proteomic studies. We describe advances in β‐elimination / Michael addition‐based approaches which allow for mass spectrometry‐based identification and comparative quantification of O‐phosphate or O‐GlcNAc‐modified peptides, as well as cysteine‐containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope‐coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O‐phosphorylation and O‐GlcNAc modification sites. Specificity of O‐phosphate vs. O‐GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O‐GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post‐translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large‐scale quantitative analysis of both protein expression and serine / threonine post‐translational modifications.

[1]  R. Scheller,et al.  Phosphorylated Syntaxin 1 Is Localized to Discrete Domains Along a Subset of Axons , 2000, The Journal of Neuroscience.

[2]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[3]  Gerald W. Hart,et al.  Glycosylation of Nucleocytoplasmic Proteins: Signal Transduction and O-GlcNAc , 2001, Science.

[4]  O. Fiehn,et al.  Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography / mass spectrometry , 2022 .

[5]  D. Forde,et al.  Mass spectrometric identification of amino acid transformations during oxidation of peptides and proteins: modifications of methionine and tyrosine. , 1995, Analytical chemistry.

[6]  Lance Wells,et al.  Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications* , 2002, Molecular & Cellular Proteomics.

[7]  O. Jensen.  Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. , 2004, Current opinion in chemical biology.

[8]  G. Voglino,et al.  Le variant βE et le code de phosphorylation des caséines bovines , 1974 .

[9]  Y. Nishizuka,et al.  Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase. , 1985, The Journal of biological chemistry.

[10]  J. Hanover.  Glycan‐dependent signaling: O‐linked N‐acetylglucosamine , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  A. Thompson,et al.  Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition. , 2003, Analytical chemistry.

[12]  J. Honnorat,et al.  Collapsin response mediator proteins (CRMPs) , 2003, Molecular Neurobiology.

[13]  G. Hart,et al.  Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952 , 1994, Journal of virology.

[14]  Rainer Cramer,et al.  Evaluation of Two-dimensional Differential Gel Electrophoresis for Proteomic Expression Analysis of a Model Breast Cancer Cell System* , 2002, Molecular & Cellular Proteomics.

[15]  F. Cross,et al.  Accurate quantitation of protein expression and site-specific phosphorylation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Storm,et al.  Identification of the protein kinase C phosphorylation site in neuromodulin. , 1990, Biochemistry.

[17]  G. Hart,et al.  Diverse regulation of protein function by O-GlcNAc: a nuclear and cytoplasmic carbohydrate post-translational modification. , 2002, Current opinion in chemical biology.

[18]  B. Chait,et al.  Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides. , 2003, Analytical chemistry.

[19]  Richard D. Smith,et al.  Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides. , 2002, Analytical chemistry.

[20]  F. H. Lopes da Silva,et al.  Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals. , 2001, Molecular biology of the cell.

[21]  D. Storm,et al.  Interactions between Neurogranin and Calmodulin in Vivo * , 1999, The Journal of Biological Chemistry.

[22]  A. Burlingame,et al.  Urinary proteomics of renal Fanconi syndrome. , 2004, Contributions to nephrology.

[23]  R. Aebersold,et al.  Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry , 2001, Nature Biotechnology.

[24]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[25]  Robert J Chalkley,et al.  Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13C-Isotope-coded Affinity Tag and Multidimensional Chromatography* , 2003, Molecular & Cellular Proteomics.

[26]  T. Hunter,et al.  Signaling—2000 and Beyond , 2000, Cell.

[27]  Matthias Mann,et al.  Mass spectrometric-based approaches in quantitative proteomics. , 2003, Methods.

[28]  L. Heilmeyer,et al.  Sequence analysis of phosphoserine‐containing peptides , 1986, FEBS letters.

[29]  R. Scheller,et al.  Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Birgit Schilling,et al.  Phosphospecific proteolysis for mapping sites of protein phosphorylation , 2003, Nature Biotechnology.

[31]  G. J. Rademaker,et al.  Mass spectrometric determination of the sites of O-glycan attachment with low picomolar sensitivity. , 1998, Analytical biochemistry.

[32]  John R Yates,et al.  Analysis of quantitative proteomic data generated via multidimensional protein identification technology. , 2002, Analytical chemistry.

[33]  K. Tomer,et al.  Detection and sequencing of phosphopeptides , 2000, Journal of the American Society for Mass Spectrometry.

[34]  T. Shirao,et al.  Clustering and anchoring mechanisms of molecular constituents of postsynaptic scaffolds in dendritic spines , 2001, Neuroscience Research.

[35]  K. Schey,et al.  Identification of tryptophan oxidation products in bovine α‐crystallin , 1998, Protein science : a publication of the Protein Society.

[36]  W. Hemmer,et al.  Functional aspects of creatine kinase in brain. , 1993, Developmental neuroscience.

[37]  Wei Li,et al.  Susceptibility of the hydroxyl groups in serine and threonine to beta-elimination/Michael addition under commonly used moderately high-temperature conditions. , 2003, Analytical biochemistry.

[38]  Richard D. Smith,et al.  Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. , 2001, Analytical chemistry.

[39]  Ruedi Aebersold,et al.  Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. , 2002, Journal of proteome research.

[40]  R. Aebersold,et al.  Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. , 2003, Current opinion in biotechnology.

[41]  J. Gebler,et al.  Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry. , 2001, Rapid communications in mass spectrometry : RCM.

[42]  P. Roepstorff,et al.  Phospho‐proteomics: Evaluation of the use of enzymatic de‐phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis , 2001, Proteomics.

[43]  J. Porath,et al.  Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. , 1986, Analytical biochemistry.

[44]  J. Baudier,et al.  Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain. , 1991, The Journal of biological chemistry.

[45]  G. Hart,et al.  Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins. , 1997, Annual review of biochemistry.

[46]  J. Deloulme,et al.  A comparative study of the distribution of α- and γ-enolase subunits in cultured rat neural cells and fibroblasts , 1997, International Journal of Developmental Neuroscience.

[47]  K. Resing,et al.  The characterization of protein post-translational modifications by mass spectrometry. , 2003, Accounts of chemical research.

[48]  G. Marino,et al.  Mapping Phosphorylation Sites: A New Strategy Based on the Use of Isotopically-Labelled Dithiothreitol and Mass Spectrometry , 2004, European journal of mass spectrometry.

[49]  Y. Mechref,et al.  Matrix-assisted laser desorption/ionization mass spectrometry compatible beta-elimination of O-linked oligosaccharides. , 2002, Rapid communications in mass spectrometry : RCM.

[50]  A Aitken,et al.  Specificity of 14-3-3 isoform dimer interactions and phosphorylation. , 2001, Biochemical Society transactions.

[51]  B. Chait,et al.  Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome , 2001, Nature Biotechnology.

[52]  J. Patrick,et al.  Neuronal localization of the cyclophilin A protein in the adult rat brain , 1996 .

[53]  A. Means,et al.  Regulatory cascades involving calmodulin-dependent protein kinases. , 2000, Molecular endocrinology.

[54]  M. Byford.  Rapid and selective modification of phosphoserine residues catalysed by Ba2+ ions for their detection during peptide microsequencing. , 1991, The Biochemical journal.