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1.  Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry 
mAbs  2012;4(1):24-44.
Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.
PMCID: PMC3338939  PMID: 22327428
host cell proteins; protein quantification; biotherapeutic proteins; mAbs; HCP
2.  Identification of secreted glycoproteins of human prostate and bladder stromal cells by comparative quantitative proteomics 
The Prostate  2009;69(1):49-61.
Functional development of the prostate is governed by stromal mesenchyme induction and epithelial response. Stromal/epithelial signaling can be mediated through direct cell-cell contact and diffusible factors and their cell surface receptors. These inducers are likely secreted or membrane-associated extracellular proteins. Given the importance of intercellular communication, it is possible that diseases like cancer could arise from a loss of this communication. One approach to gain a molecular understanding of stromal cells is to identify, as a first step, secreted stromal signaling factors. We proposed to do this by comparative analysis between bladder and prostate.
Secreted proteins were identified from cultured normal prostate and bladder stromal mesenchyme cells by glycopeptide-capture method followed by mass spectrometry. Differences in protein abundance between prostate and bladder were quantified from calculated peptide ion current area (PICA) followed by Western validation. Functional and pathway analyses of the proteins were carried out by Gene Ontology (GO) and Teranode software.
This analysis produced a list of 116 prostate and 84 bladder secreted glycoproteins with ProteinProphet probability scores ≥ 0.9. Stromal proteins upregulated in the prostate include cathepsin L, follistatin-related protein, neuroendocrine convertase, tumor necrosis factor receptor, and others that are known to be involved in signal transduction, extracellular matrix interaction, differentiation and transport.
We have identified a number of potential proteins for stromal signaling and bladder or prostate differentiation program. The prostate stromal/epithelial signaling may be accomplished through activation of the ECM-receptor interaction, complement and coagulation cascades, focal adhesion and cell adhesion pathways.
PMCID: PMC4281891  PMID: 18792917
bladder; prostate; stromal cells; secreted proteins
3.  Characterization of a Noncovalent Lipocalin Complex by Liquid Chromatography/Electrospray Ionization Mass Spectrometry 
Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin–ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound (“siderophore”) synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.
PMCID: PMC2291690  PMID: 15331587
Noncovalent complexes; lipocalin; siderocalin; liquid chromatography; electrospray ionization mass spectrometry
4.  Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5 
Biochemistry  2012;51(47):9488-9500.
Cytochrome b5 (cyt b5) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached on the underlying mechanism of cyt b5 modulation of CYP catalysis. Both cyt b5 and apo b5, are reported to stimulate the activity of several P450 isoforms. In the present study, the surface interactions of both holo and apo b5 with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the interaction models of holo/apo b5 with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b5 and apo b5 were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B’ loop of CYP3A4, a substrate recognition site (SRS). Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127 and Lys421) are of functional importance. Mutation of these residues reduced or abolished cyt b5 binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b5 and/or cytochrome P450 reductase (CPR) was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that the apo cyt b5 can dock with CYP3A4 in a manner analogous to holo cyt b5 so electron transfer from cyt b5 is not required for its effects.
PMCID: PMC3568533  PMID: 23150942
CYP3A4; cyt b5; apo b5; interaction; chemical cross-linking; MS; mutagenesis; PDB: 1TQN; 1CYO; 1I87
5.  Minimize the Detection of False Positives by the Software Program DetectShift for 18O-Labeled Cross-Linked Peptide Analysis 
In order to speed up the process of cross-linked peptide identification and characterization, we have previously reported the development of Pro-CrossLink 1, a suite of software tools consisting of three programs, DetectShift, IdentifyXLink, and AssignXLink for mass spectrometric data analysis. Since its public disclosure, Pro-CrossLink has been downloaded by 101 research groups. Pro-CrossLink users have provided us with valuable feedback on the use of the program DetectShift. Here we assess some reasons for the generation of false positives by DetectShift. In addition, we provide users with suggestions on optimal parameter setting and efficient use of the software program.
PMCID: PMC2749487  PMID: 19023145
6.  Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents 
Journal of neurochemistry  2004;91(2):438-450.
We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.
PMCID: PMC2747775  PMID: 15447677
mass spectrometry; metabotropic glutamate receptor; protein interaction; proteomics
7.  Human p53 is Inhibited by Glutathionylation of Cysteines Present in the Proximal DNA-Binding Domain During Oxidative Stress† 
Biochemistry  2007;46(26):7765-7780.
The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA-binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and reversible posttranslational modification, namely, S-glutathionylation in stressed states including DNA damage. First, a rapid and direct incorporation of biotinylated GSH or GSSG into the purified recombinant p53 protein was observed. The modified p53 had significantly decreased ability to bind its consensus DNA sequence. Reciprocal immunoprecipitations and a GST-overlay assay showed that p53 in tumor cells was marginally glutathionylated, however, the modification increased greatly after oxidant and DNA-damaging treatments. GSH-modification coexisted with the serine phophorylations in activated p53, and the thiol-conjugated protein was present in nuclei. When tumor cells treated with camptothecin or cisplatin were subsequently exposed to glutathione-enhancing agents, p53 underwent dethiolation accompanied by detectable increases in p21waf1 expression, relative to the DNA damaging drugs alone. Mass spectrometry of GSH-modified p53 protein identified the cysteines 124, 141 and 182, all present in the proximal DNA-binding domain, as the sites of glutathionylation. Biotinylated maleimide also reacted rapidly with Cys141, implying this to be the most reactive cysteine on p53 surface. The glutathionylatable cysteines were found to exist in a negatively-charged microenvironment in cellular p53. Molecular modeling studies located Cys124 and 141 to the dimer interface of p53 and showed glutathionylation of either residue would inhibit p53-DNA association, and also interfere with protein dimerization. These results show for the first time that shielding of reactive cysteines contributes to a negative regulation for human p53, and imply that such an inactivation of the transcription factor may represent an acute defensive response with significant consequences for oncogenesis.
PMCID: PMC2518322  PMID: 17555331
8.  Comparison of ESI MS Interfaces for the Analysis of UV-crosslinked Peptide-Nucleic Acid Complexes 
In this report, the effectiveness of high performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry (ESI-MS) is examined as a tool for identifying the sites of crosslinking in a protein that has been photoreacted with a non-photolabeled oligonucleotide. ESI-MS and MALDI-MS analyses preceded by off-line microflow and nanoflow HPLC, on-line microflow HPLC/ESI, and on-line nanoflow HPLC/ESI interfaces were performed in order to determine their relative effectiveness in separating mixtures of nucleopeptides and identifying sites of crosslinking on the individual components. The characteristics of these four techniques as well as possibilities for improving the analysis of nucleopeptides by ESI MS are compared and discussed.
PMCID: PMC2238692  PMID: 18042442
9.  The 64-Kilodalton Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails 
Journal of Virology  2003;77(4):2377-2384.
The filamentous virion of the closterovirus Beet yellows virus (BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography-tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement.
PMCID: PMC141117  PMID: 12551975

Results 1-9 (9)