Robert J. Chalkley

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis. In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis. In recent years, the emphasis within the proteomics field has moved from the identification of isolated proteins (1Clauser K.R. Hall S.C. Smith D.M. Webb J.W. Andrews L.E. Tran H.M. Epstein L.B. Burlingame A.L. 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In the last few years, enzymatic digestion of unseparated protein mixtures followed by separation of peptides by multidimensional liquid chromatography (LC) 1The abbreviations used are: LCliquid chromatographyICATisotope-coded affinity tagMSmass spectrometryMS/MStandem mass spectrometryESIelectrospray ionizationMALDImatrix-assisted laser desorption/ionizationACNacetonitrilePrPprion proteinSCXstrong has been used as an of the proteome by multidimensional protein identification Scholar) peptides to proteins in Saccharomyces using this this approach to peptide separation with the and of mass spectrometric A in the that of peptides to protein is L. S.C. Baldwin M.A. Burlingame A.L. of the from using mass spectrometry and new Biol. Chem. Scholar). liquid chromatography isotope-coded affinity tag mass spectrometry tandem mass spectrometry electrospray ionization matrix-assisted laser desorption/ionization prion protein time-of-flight cleavable ICAT human for characterizing changes in complex mixtures been using of these two-dimensional samples on separate and protein with the use of R.D. M. C. of two-dimensional gel by and 1: Scholar). in protein can to to of protein on two-dimensional an as a of a large of manual to the in a B. A. and of two two-dimensional electrophoresis and 2002; Scholar). approach that has many of the to is differential gel electrophoresis M. gel a gel method for changes in protein Scholar, S. P. A. of two-dimensional differential gel electrophoresis for expression of a cancer Cell. Proteomics. 2002; 1: Scholar). spectrometry is a technique as are highly on the and of the combined with has been used for many to of A. in with mass 1999; Scholar, J.E. of and using Scholar), this has been to peptides Smith spectrometric of proteins: with Chem. Scholar) and proteins L. L. A for the in vivo of levels in by mass Scholar, A. S. P. of peptides for and sequence Scholar, A. J. C. for with two of Chem. Scholar, L. N. Y. Smith R.D. peptide by and Chem. 2002; Scholar, Martin L.E. L.B. A. of the by using peptides and peptide Chem. 2003; Scholar). The of isotope-coded affinity tag for and a in sample S. A. N. Protein by mass spectrometry and sequence for cancer in the 1999; 20: Scholar). of i) an that with the of a ii) a to of peptides by avidin affinity and a that the ICAT this and a in mass of and In a sample with reagent and the with of ICAT samples are combined and the components are affinity purified by of the mass spectrometric the mass for of differing in mass by and relative of proteins by of the integrated of the two The of peptides of by tandem to a from the of the protein by the of this several of this reagent were i) the and peptides by S. of peptides in Chem. ii) the tag and of peptides produced many in the to the tag the peptide Smith R.D. of Chem. 2002; the mass addition from the of the tag the of peptides the for by and the choice of mass for the ICAT reagent produced peptides containing two ICAT-labeled and the of a of has been The of these used ICAT on and a H. proteome by and mass 2002; 20: Scholar). of the peptides followed by of this to the ICAT the use of as the and from the separation of and the use of a ICAT reagent that an the with the reactive In this ICAT and peptide affinity is followed by in of the The of this are the addition of a to the and in the of from using as the this reagent employs 13C as the for the the and peptides by to and the In most ICAT B. J. of proteins using multidimensional chromatography and isotope-coded affinity 2002; 1: Scholar, H. B. of a liquid chromatography for proteome Chem. 2002; Scholar, J. H. profiling of proteins using isotope-coded affinity and mass Scholar, B. H. Toward a approach to protein identification by mass spectrometry.J. Scholar), sample has been a of protein used in these from to to the to sample with the of many protein samples of in sample losses during the ICAT volatile that by were in ICAT peptides are these peptides been for separation and identification by multidimensional and approach to the differential profiling strength provided by the ICAT strategy with the high sequence coverage by multidimensional the power of this from two of that high on and i) the characterization of proteins to the murine prion and ii) the characterization of proteins from human tracheal epithelium gland secretions. were using two different a with electrospray ionization for and an Proteomics matrix-assisted laser desorption/ionization with of separated The is a time-of-flight the latter is an a of on the of number of peptides number of proteins and proteins and by cleavable ICAT (cICAT) identified in the of the avidin chromatography. the of mass in and in sample and of ICAT and and were from and and peptides and proteins were from from were with and to The A and were from experimental avidin by from and from The used for containing from of from from to were to and were from and were from and to the protocol with the ICAT The complex protein samples were with the cleavable reagent using a protein samples were in with to for ICAT were in and for were combined and to the of to digestion by the addition of of and to for chromatography used to from the tryptic peptides and to peptide of the samples were to and by the addition of using a with an with and a using multiple sample a A with a A of and of A with A separation employed from to for sample and of followed by a of from from and the with a of in A. were in The were by the addition of of using as to the up to The for the avidin affinity chromatography. 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Scholar). were using on a mass and by on a the latter samples were analyzed separation from targets using a on were to and combined in are the of the peptide in the and with these two different peptide in the of the identification of proteins that with the prion protein and in is a peptide from The is of the two mass spectrometric is the of using the for an in the In for the of of in the large of are to high by as by the in and and protein identification confidence for of low the is for a few to with the the number of to on the as the and of to the for the for the peptides and coverage for mouse peptides identified by are in peptides identified by are and identified by are is a the number of for and the of the to is by using on the mass many are for from a the peptides been the peptides are in the and different are for analysis. 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Scholar), and a of that is in the The in and of the 1998; Scholar). of the that the identification of are in a and peptides were identified as to this with being of these were identified using of the mass were to were to the sequence coverage that by the identification of proteins in the of prion this a mouse employed that levels of a on a had been that this point mice to with J. J. P. of prion in Natl. Acad. Sci. U. S. A. 2002; Scholar). The of samples from and mice significant in the of the identified proteins to the of identified proteins and are and of the proteins identified in the changes in abundance as the and on this the of multidimensional chromatography and significant multidimensional chromatography in of proteins in the of prion of these S. S. and M. in In a the changes in the proteome of the human in a patient with cystic in order to of in these from the of a cystic patient from the of a that were for used as a of the and of the tracheal gland were from the and of using the to from a large number of proteins A of the proteins identified the number of peptides the mass of is in and is in the proteins to a high based on the number of peptides human and several of In several proteins were identified that are to low as and proteins and an of sequence coverage the proteins, peptide sequence coverage of the the ICAT coverage of the peptides for of with the tryptic peptides and being with the the of this to very the protein peptides were in the avidin that were peptide the peptide the peptide several proteins were identified on the of peptides were identified for these of these the cystic identified on the of the peptide and for this peptide are in The used for the of this protein as D.A. and Biol. 1999; Scholar), in the cystic were with previous that increased levels in as cystic in Rev. 2002; Scholar, J. 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A. and A. in the of the large number of that were in this employed and an of Protein the were for using a by with the The that of the were for for Protein were by in the to used to a that on the of a In the of Protein employed a new to from that had been within the the on the of of were combined for and the and as a combined the identification of the of the for the components in the protein identifications the different the with confidence in their by multiple and of this a strategy for the content and relative of proteins two strategy the new reagent to the information and multidimensional chromatography to improve protein identification confidence and to proteome A of this technique is the to highly separation of nonlabeled peptides using the affinity to for a of this of the use of 13C as the in of the tag the addition of is peptides containing two and a peptide containing the of of the the tag is and under used for The components for the characterization of samples are the of sample of sample of the mass spectrometric and the of the and analysis. in this of sample were for of the technique were sample to samples that were compatible with and the of high tandem mass spectrometric analysis. While the two in this were very samples high complexity, low protein and were to from their is established that separation of protein the number of peptides that can identified in complex samples by M.A. Medzihradszky K.F. B. Burlingame A.L. laser desorption/ionization with time-of-flight mass spectrometry for protein and Chem. Scholar). In peptides of low abundance low ionization are identified in separated electrospray ionization to of Chem. Scholar, C. 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Protein O-GlcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GlcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GlcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment--significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GlcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GlcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GlcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GlcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification.