We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-g (Ϸ600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.yeast phosphoproteome ͉ network analysis I n an earlier study of the yeast phosphoproteome (1), we digested proteins from a whole cell lysate with trypsin, used immobilized metal-affinity chromatography (IMAC) to enrich the sample for phosphopeptides, and analyzed the resulting mixture by nano-flow HPLC interfaced to electrospray ionization tandem mass spectrometry (MS/MS). Low-energy collision-activated dissociation (CAD) was used to fragment the peptide backbone and to produce ions of types b and y (Fig. 1) required for successful sequence analysis and identification of phosphorylation sites. We detected Ͼ1,000 phosphopeptides but defined only 383 sites of phosphorylation, largely because the CAD process often promoted elimination of phosphoric acid from Ser and Thr residues without breaking the amide bonds along the peptide backbone. The resulting MS/MS spectra were essentially devoid of sequence information.To circumvent this problem, we have modified the LTQ mass spectrometer for ion/ion chemistry and now fragment both peptides (2) and intact proteins (3) by electron transfer dissociation (ETD). In this process, fluoranthene radical-anions are generated in a chemical ionization source and used as reagents to transfer an electron to a multiply charged peptide generated by electrospray ionization. This reaction is highly exothermic, reduces the peptide charge by one, and triggers fragmentation of the peptide backbone to produce a homologous series of complementary fragment ions of type c and z⅐ (Fig. 1). Subtraction of m/z values for fragments within a given ion series that differ by a single amino acid affords the mass...

We describe the design and performance of a prototype high performance hybrid mass spectrometer. This instrument consists of a linear quadrupole ion trap (QLT) coupled to a Fourier transform ion cyclotron resonance mass analyzer (FTMS). This configuration provides rapid and automated MS and MS/MS analyses, similar to the "data dependent scanning" found on standard 3-D Paul traps, but with substantially improved internal scan dynamic range, mass measurement accuracy, mass resolution, and detection limits. Sequence analysis of peptides at the zeptomole level is described. The recently released, commercial version of this instrument operates in the LC/MS mode (1 s/scan) with a mass resolution of 100 000 and is equipped with automatic gain control to provide mass measurement accuracy of 1-2 ppm without internal standard. Methodology is described that uses this instrument to compare the post-translational modifications present on histone H3 isolated from asynchronously growing cells and cells arrested in mitosis.

Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.epigenetics | kinetochore | mass spectrometry

Leukemias are highly immunogenic but have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these post-translationally modified neoantigens provide logical targets for cancer immunotherapy.

In this investigation of developmental changes in the coordination of perceived optical flow and postural responses, 4 age groups of infants (5-, 7-, 9-, and 13-month-olds) were tested while seated on a force plate in a "moving room." During each trial the walls oscillated in an anteroposterior direction for 12 s, and the postural sway of the infant was measured. The results revealed that infants perceived the frequency and amplitude of the optical flow and scaled their postural responses to the visual information. This scaling was present even before infants could sit without support, but it showed considerable improvement during the period when infants learn to sit. Taken together, these results suggest that the visuomotor coordination necessary for controlling sitting is functional prior to the onset of independent sitting but becomes more finely tuned with experience.