Biomedical subjects

Yingda Xu

Publications and source records attributed to Yingda Xu.

7 recordsLinked to original sources

PubMed2007

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS.

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.

Animals↗

PubMed2006

14-3-3gamma binds to MDMX that is phosphorylated by UV-activated Chk1, resulting in p53 activation.

It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14-3-3gamma and Chk1. 14-3-3gamma was identified as an MDMX-associated protein via an immuno-affinity purification-coupled mass spectrometry. Consistently, 14-3-3gamma directly interacted with MDMX in vitro, and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild-type, but not the kinase-dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14-3-3gamma-MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN-01 repressed all of these effects. Moreover, overexpression of 14-3-3gamma, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX-enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14-3-3gamma by siRNA reduced UV-induced p53 level and G1 arrest. Thus, these results demonstrate 14-3-3gamma and Chk1 as two novel regulators of MDMX in response to UV irradiation.

14-3-3 Proteins↗

PubMed2006

Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.

Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.

Acetylation↗

PubMed2006

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea.

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.

Amino Acid Sequence↗

PubMed2004

Use of polymer-modified MALDI-MS probes to improve analyses of protein digests and DNA.

The use of sample probe surfaces patterned with 200-microm-diameter spots of hydrophilic, charged polymers significantly enhances the analysis of protein digests and DNA by MALDI-MS. Selective adsorption on these polymer-modified surfaces allows collection of specific proteolytic peptides, while subsequent rinsing of the deposited sample removes contaminants. In the case of partially digested myoglobin, the mass spectrum obtained using a sample probe modified with polyanionic functionalities permits detection of 22 proteolytic fragments, while analysis using a stainless steel MALDI sample probe gives only 11 detectable fragments. Similarly, during the analysis of bovine serum albumin digests, the use of several different surface-modified MALDI sample probes increases sequence coverage from 61.3 to 74.5%. Detection of phosphorylated peptides can be quite challenging during analyses of phosphoprotein digests by MALDI-MS because these anionic proteolytic fragments have low ionization efficiencies. However, MALDI signals from the phosphorylated proteolytic fragments sometimes increase dramatically when using a sample probe surface modified by a polycation (polyethylenimine or poly(acrylic acid) complexed with Fe(3+)). The signal enhancement apparently occurs because the positive surface selectively binds the phosphorylated peptides. The use of patterned, polycationic surfaces also shows great promise for selective adsorption and decontamination of DNA samples; a simple water rinse diminishes or eliminates the formation of multi-ion adducts, thereby improving mass resolution during subsequent analysis by MALDI-MS.

Acrylic Resins↗

PubMed2003

Patterned monolayer/polymer films for analysis of dilute or salt-contaminated protein samples by MALDI-MS.

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-microm diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance FT-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.

Acrylic Resins↗

PubMedDate not supplied

Non-specific, on-probe cleanup methods for MALDI-MS samples.

High concentrations of contaminants such as salts and surfactants are often present in biological samples to solubilize or stabilize analytes such as proteins. Unfortunately, the presence of those contaminants often precludes direct analysis by MALDI-MS. Selective adsorption of analytes directly on modified MALDI probes, followed by rinsing to remove contaminants, overcomes this problem. This review focuses on various modifications of MALDI probes to allow the adsorption of proteins and DNA, even in a large excess of salt or surfactant. Interfaces deposited on the MALDI probes to adsorb analytes include films of commercial polymers, thin layers of matrix crystals, self-assembled monolayers, and ultrathin polymer films. Hydrophobic and ionic interactions both effect analyte adsorption on those interfaces, and patterned interfaces allow the concentration and purification of analyte molecules.

Drug Contamination↗