Biomedical subjects

Fanyu Meng

Publications and source records attributed to Fanyu Meng.

9 recordsLinked to original sources

PubMed2025

A Functionally Conserved yet Dynamically Evolving Toolkit Underpinning Molluscan Biomineralization: Insights From Shell and Radula.

The molluscan shell and radula constitute pivotal molluscan innovations, each characterized by distinct functions and diverse forms, regulated by the highly specific biomineralization regulatory networks. Despite their paramount importance, the conserved components and adaptive evolutionary processes governing these regulatory networks remain unresolved. To address this knowledge gap, we advocate for the integration of data from less-explored lineages, such as Scaphopoda, as an essential step. This study presents the inaugural comprehensive transcriptome analysis of Pictodentalium vernedei, a representative species of Scaphopoda distinguished by a unique and evolutionarily conserved shell morphology and radula structure. Furthermore, comparative transcriptome/genome analyses are employed to unravel the conservatism and evolutionary innovation of the involved biomineralization regulatory elements. Our findings underscore the central role of secretomes in governing biomineralization processes, and we identified a fundamental set of 26 domains within molluscan secretomes, forming an essential functional protein domain repertoire necessary for the transformation of inorganic ions into biomineralized structures. This core biomineralization toolkit has undergone independent expansion and lineage-specific recruitment, giving rise to novel, modular domain architectures. This may be essential for the functional specialization and morphological diversification of shell and radula structures. These evolutionary processes are driven by the independent co-option of ancient genes and the emergence of novel de novo genes. This comprehensive investigation not only contributes insights into the evolution of molluscan biomineralization structures but also establishes avenues for further scholarly exploration.

Animals↗

PubMed2006

Quantitative analysis of complex peptide mixtures using FTMS and differential mass spectrometry.

Label-free LC-MS profiling is a powerful quantitative proteomic method to study relative peptide abundances between two or more biological samples. Here we demonstrate the use of a previously described comparative LC-MS method, differential mass spectrometry (dMS), to analyze high-resolution Fourier transform mass spectrometry (FTMS) data for detection and quantification of known peptide differences between two sets of complex mixtures. Six standard peptides were spiked into a processed plasma background at fixed ratios from 1.25:1 to 4:1 to make two sets of samples. The resulting mixtures were analyzed by microcapillary LC-FTMS and dMS. dMS successfully identified five out of the six peptides as statistically significant differences (p <or= 0.005). In this experiment, the smallest fold change reliably detected by our method was 1.5:1, and the errors of estimated ratios of concentrations were less than 20% for peptides spiked at 1.5:1 to 4:1. We conclude that LC-FTMS coupled with dMS is a useful label-free quantitative MS method that can be used to detect subtle yet statistically significant peptide differences in complex protein mixtures, including plasma samples.

Amino Acid Sequence↗

PubMed2004

Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics.

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.

Acetylation↗

PubMed2003

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.

Acetylation↗

PubMed2003

Web and database software for identification of intact proteins using "top down" mass spectrometry.

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

Amino Acid Sequence↗

PubMed2002

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry.

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.

Algorithms↗

PubMed2002

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.

Archaeal Proteins↗

PubMedDate not supplied

Detection and localization of protein modifications by high resolution tandem mass spectrometry.

For interrogation of peptides with diverse modifications, no other instrument is as versatile as the Fourier-transform mass spectrometer (FTMS). Particularly using electrospray ionization (ESI), many intact proteins and their proteolytic products harboring post-translational and chemical modifications (PTMs) have been studied by high resolution tandem mass spectrometry (MS/MS). The widely touted analytical figures of merit for FTMS in fact have translated into clarity when analyzing PTMs from phosphorylations to disulfides, oxidations, methylations, acetylations, and even exotic PTMs found in the biosynthesis of antibiotics and other natural products. A top down approach to PTM detection and localization is proving extensible to an increasing variety of PTMs, some of which are stable to MS/MS at the protein level but unstable to amide bond cleavage by threshold dissociations at the level of small peptides <3 kDa. In contrast, MS/MS using electron capture dissociation (ECD) allows precise localization of even labile PTMs given enough sample and abundant molecular ions. Finally, this brief synopsis of recent literature highlights specific PTMs that perturb the protein backbone therefore altering MS/MS fragmentation patterns. Thus, FTMS will continue its expansion into more laboratories in part because of its ability to detect and deconvolute the regulatory mechanisms of biology written in the language of PTMs.

Amino Acid Sequence↗

PubMedDate not supplied

Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS.

Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.

Amino Acid Sequence↗