A label-free mass spectrometry method for relative quantitation of ß-tubulin isotype expression in human tumor tissue.

PURPOSE: Quantitation of ß-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of ß-tubulin isotypes. EXPERIMENTAL DESIGN: Using isotype-specific reporter peptides, we determined relative ß-tubulin isotype expression levels in human lung tumor tissue. RESULTS: Four reporter peptides were chosen to quantitate the ßI/ßII, ßIV, ßIII, and ßV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine ß-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low ßIII-tubulin expressing using immunohistochemistry. It was found that ßI/ßII (accounting for 18.7-65.7% of total ß-tubulin) and ßIVa/ßIVb (26.3-79.1%) were the most abundant isotypes and that the ßIII (0-8.9%) and ßV (1.0-10.4%) were less abundant in the tissue. We also categorized the samples as high or low ßIII-tubulin expressing. CONCLUSION AND CLINICAL RELEVANCE: With this method we can determine the relative expression levels of ß-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.

Structural evidence for cooperative microtubule stabilization by Taxol and the endogenous dynamics regulator MAP4.

Microtubules (MTs) composed of αß-tubulin heterodimers are highly dynamic polymers, whose stability can be regulated by numerous endogenous and exogenous factors. Both the antimitotic drug Taxol and microtubule-associated proteins (MAPs) stabilize this dynamicity by binding to and altering the conformation of MTs. In the current study, amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to examine the structural and dynamic properties of the MT complex with the microtubule binding domain of MAP4 (MTB-MAP4) in the presence and absence of Taxol. The changes in the HDX levels indicate that MTB-MAP4 may bind to both the outside and the luminal surfaces of the MTs and that Taxol reduces both of these interactions. The MTB-MAP4 binding induces conformational rearrangements of α- and ß-tubulin that promote an overall stabilization of MTs. Paradoxically, despite Taxol's negative effects on MAP4 interactions with the MTs, its binding to the MTB-MAP4-MT complex further reduces the overall deuterium incorporation, suggesting that a more stable complex is formed in the presence of the drug.

Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii.

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.

IVICAT for the masses: an improved technique for permethylation of peptides.

To determine the levels of post-translational modifications, we needed a quantitative technique that would allow comparison of the amounts of acetylated versus mono-, di-, and tri-methylated lysines in histones. One method, IVICAT, generates trimethyl-amines and could be used, but is technically challenging. We have modified this technique to be used with standard laboratory equipment so that this chemistry is accessible to most proteomics laboratories.

Nonprotein based enrichment method to analyze peptide cross-linking in protein complexes.

Cross-linking analysis of protein complexes and structures by tandem mass spectrometry (MS/MS) has advantages in speed, sensitivity, specificity, and the capability of handling complicated protein assemblies. However, detection and accurate assignment of the cross-linked peptides are often challenging due to their low abundance and complicated fragmentation behavior in collision-induced dissociation (CID). To simplify the MS analysis and improve the signal-to-noise ratio of the cross-linked peptides, we developed a novel peptide enrichment strategy that utilizes a cross-linker with a cryptic thiol group and using beads modified with a photocleavable cross-linker. The functional cross-linkers were designed to react with the primary amino groups in proteins. Human serum albumin was used as a model protein to detect intra- and intermolecular cross-linkages. Use of this protein-free selective retrieval method eliminates the contamination that can result from avidin-biotin based retrieval systems and simplifies data analysis. These features may make the method suitable to investigate protein-protein interactions in biological samples.