Fanyu Meng

Informatics and multiplexing of intact protein identification in bacteria and the archaea.

Although direct fragmentation of protein ions in a mass spectrometer is far more efficient than exhaustive mapping of 1–3 kDa peptides for complete characterization of primary structures predicted from sequenced genomes, the development of this approach is still in its infancy. Here we describe a statistical model (good to within ∼5%) that shows that the database search specificity of this method requires only three of four fragment ions to match (at ±0.1 Da) for a 99.8% probability of being correct in a database of 5,000 protein forms. Software developed for automated processing of protein ion fragmentation data and for probability-based retrieval of whole proteins is illustrated by identification of 18 archaeal and bacterial proteins with simultaneous mass-spectrometric (MS) mapping of their entire primary structures. Dissociation of two or three proteins at once for such identifications in parallel is also demonstrated, along with retention and exact localization of a phosphorylated serine residue through the fragmentation process. These conceptual and technical advances should assist future processing of whole proteins in a higher throughput format for more robust detection of co- and post-translational modifications.

Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a “histone code” for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc/HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3/H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing Factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms.

Fourier-transform mass spectrometry for automated fragmentation and identification of 5-20 kDa proteins in mixtures

When presented with a mixture of intact proteins, electrospray ionization with Fourier‐transform mass spectrometry (ESI‐FTMS) has the capability to obtain direct fragmentation information from isolated ions. However, the automation of this capability has not been achieved to date. We have developed software for unattended acquisition of protein tandem mass spectrometry (MS/MS) data and batch processing of the resulting files for identification of whole proteins. Mixtures of both protein standards (8–29 kDa) and Methanococcus jannaschii cytosolic proteins (up to six components + 20 kDa) were infused via an autosampler, and MS/MS data were acquired without human intervention. The acquisition software recognizes ESI charge state patterns, generates protein‐specific isolation waveforms on‐the‐fly, and fragments ions using two different infrared laser times. In addition to protein standards, five wild‐type proteins (7–14 kDa) were identified automatically with 100% sequence coverage from the M. jannaschii database. The software underpins a measurement platform for sample‐dependent acquisition of MS/MS data for whole proteins, a critical step to realize proteomics with 100% sequence coverage in a higher throughput setting.