SUMMARY Lysine methylation occurs on both histone and non-histone proteins. However, our knowledge on the prevalence and function of non-histone protein methylation is poor. We describe here an approach that combines peptide array, bioinformatic and mass spectrometric analyses to systematically identify lysine methylation sites in proteins and methyllysine-mediated protein-protein interactions. We demonstrate the utility of this approach by identifying a methyllysine-driven interactome of the heterochromatin protein (HP) 1β and uncovering, simultaneously, numerous methyllysine sites on non-histone proteins. The HP1β interactome is enriched with proteins involved in DNA damage repair and RNA splicing. We showed that lysine methylation played a pivotal role in the function of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and its interaction with HP1β during DNA damage response. Moreover, by combining heavy methyl SILAC with Multiple Reaction Monitoring (MRM) mass spectrometry (MS), we showed that lysine methylation underwent widespread and large changes in response to DNA damage. Our work indicates that lysine methylation is a highly dynamic post-translational modification occurring frequently on non-histone proteins and that the approach presented herein may be extended to many methyllysine-binding modules to systematically uncover lysine methylation events in the cell.

The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometrybased method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Human embryonic stem cells (hESCs)1 are non-transformed cell lines that can proliferate indefinitely in culture, although maintaining the potential to form all primary human cell types (pluripotency) (1, 2). These cells, which originate from the inner cell mass of pre-implantation blastocysts, represent a unique source of human cells for cell replacement therapies and for creating model human systems for understanding disease and development (3). Like other mammalian ESCs, hESCs were originally derived and propagated on replication-deficient mouse embryonic fibroblast (MEF) feeder cells in serum (2, 4), with varying efficiencies (5). At the heart of this variability is a lack of understanding of the regulatory pathways and growth factors that govern hESC self-renewal and pluripotency (6). This ambiguity restricts the application of hESCs in both research and therapeutic applications.We hypothesize that under optimal hESC culture conditions, there exist autocrine and paracrine growth factors, produced both by the feeder cells and the hESCs themselves, that establish the complex microenvironment required to retain hESC potential in culture. Previous genomic-based studies suggested the presence of such networks of hESC transcriptional regulation (7); however, these networks were not correlated to the extracellular microenvironment that ultimately controls hESC fate. Moreover, prior attempts to identify proteins within the hESC microenvironment using MSbased approaches produced few potential candidate regulators and provided little new insight or tangible improvements upon hESC line derivation and culture (6, 8 -11).From the ‡Don Rix Protein Identification Facility,

Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b 6 /f (Cyt b 6 /f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b 6 /f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700 + indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b 6 /f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b 6 /f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins.

Background:The role of flagellin glycosylation is not well understood. Results:The Burkholderia cenocepacia flagellin is glycosylated on at least 10 different sites. Conclusion:The presence of glycan in flagellin significantly impaired the inflammatory response of epithelial cells. Significance: Flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis. It relies on several virulence factors for host colonization, including glycosylated flagella. C. jejuni NCTC 11168 modifies its flagellins with pseudaminic acid derivatives. It is also presumed to modify these proteins with legionaminic acid, although no glycopeptide evidence was available at the onset of this study. The enzyme encoded by cj1319 can be used to make legionaminic acid in vitro, but the pathway for legionaminic acid synthesis partially inferred by knockout mutagenesis in Campylobacter coli VC167 excludes Cj1319. To address this contradiction, we examined the presence of legionaminic acid in flagellin glycopeptides of wild-type (WT) C. jejuni NCTC 11168 and of a cj1319 knockout mutant. We used high-energy collision-induced dissociation to obtain amino acid sequences while also visualizing signature sugar oxonium ions. Data analysis was performed with PEAKS software, and spectra were manually inspected for glycopeptide determination and verification. We showed that legionaminic acid is present on the flagellins of C. jejuni NCTC 11168 and that flagellin glycosylation is highly heterogeneous, with up to six different sugars singly present at a given site. We found that the cj1319 mutant produces more legionaminic acid than WT, thus excluding the requirement for Cj1319 for legionaminic acid synthesis. We also showed that this mutant has enhanced chicken colonization compared with WT, which may in part be attributed to the high content of legionaminic acid on its flagella.