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.

Frozen tissue can provide reproducible proteomic results of subcellular fractionation.

Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI(N)) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI(N) values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.

Gel-based proteomics of liver cancer progression in rat.

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.

Methods in tubulin proteomics.

New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects of drugs on microtubules. The identification of tubulin isotypes and of regions of the microtubule involved in drug interactions has been advanced by proteomic methodologies. The diversity of tubulin sequences and posttranslational modifications (PTMs) can generate a complex mixture of heterodimers with unique molecular dynamics driving specific functions. Mass spectrometry (MS)-based approaches have been developed, and in combination with chromatographic and/or electrophoretic separation of tubulin polypeptides or peptides, they have contributed to our understanding of tubulin proteomics. We present protocols that we have used for the analysis of tubulin isotypes and PTMs present in tubulin isolated from cells in culture or tissues and for the identification of tubulin regions altered by microtubule-stabilizing agents. Tubulin proteomics complements structural and computer modeling information for a high-resolution view of microtubule dynamics and its alteration by drugs. These methodologies will help in providing insights into tubulin isotype-specific functions and in the design of drugs targeting either all tubulin heterodimers indiscriminately or only those containing specific isotypes.

Increased levels of a unique post-translationally modified betaIVb-tubulin isotype in liver cancer.

Identifying changes at the molecular level during the development of hepatocellular carcinoma is important for the detection and treatment of the disease. The characteristic structural reorganization of preneoplastic cells may involve changes in the microtubule cytoskeleton. Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, vesicle transport, and motility. They are comprised of multiple isotypes of alpha- and beta-tubulin. Changes in the levels of these isotypes may affect not only microtubule stability and sensitivity to drugs but also interactions with endogenous proteins. We employed a rat liver cancer model that progresses through stages similar to those of human liver cancer, including metastasis to the lung, to identify changes in the tubulin cytoskeleton during carcinogenesis. Tubulin isotypes in both liver and lung tissue were purified and subsequently separated by isoelectric focusing electrophoresis. The C-terminal isotype-defining region from each tubulin was obtained by cyanogen bromide cleavage and identified by mass spectrometry. A novel post-translational modification of betaIVb-tubulin in which two hydrophobic residues are proteolyzed from the C-terminus, thus exposing a charged glutamic acid residue, was identified. The unique form of betaIVb-tubulin was quantified in the liver tissue of all carcinoma stages and found to be approximately 3-fold more abundant in nodular and tumor tissue than in control tissue. The level of this form was also found to be increased in lung tissue with liver metastasis. This modification alters the C-terminal domain of one of the most abundant beta-tubulin isotypes in the liver and therefore may affect the interactions of microtubules with endogenous proteins.

The dehydratase activity of lacticin 481 synthetase is highly processive.

Lacticin 481 synthetase (LctM) is a bifunctional enzyme that undertakes dehydration and cyclization in the structural region of the pre-lacticin peptide (LctA) to introduce three thioether rings and one dehydrobutyrine residue. The order and timing of these events has been investigated employing high-resolution ESI-FTMS-based tandem MS/MS techniques and chemical derivatization. LctM demonstrates highly processive behavior as seen by MS analysis of the reaction course of dehydration. Furthermore, cyclization is not tightly coupled to dehydration and follows at a later stage of the enzymatic reaction.

Lacticin 481 synthetase phosphorylates its substrate during lantibiotic production.

Lacticin 481 synthetase (LctM) catalyzes the ATP-dependent conversion of a ribosomally synthesized peptide to a polycyclic thioether antibiotic. It is a bifunctional enzyme that dehydrates four Ser/Thr residues to the corresponding dehydro amino acids and catalyzes the conjugate addition of Cys residues to these dehydro residues in a regio- and stereoselective process. We show here that incubation of truncated substrates with LctM results in products that are phosphorylated in the region of dehydration. Furthermore, synthetic peptides containing phosphorylated Ser and/or Thr residues are accepted by the enzyme as substrates resulting in the elimination of phosphate and dehydro amino acid production. This activity is only observed if ADP is added as cosubstrate. These results argue strongly that the enzyme utilizes ATP to phosphorylate the Ser/Thr residues that are targeted for dehydration. ATP does not appear to be required for peptide translocation or cyclization.

Parallel interrogation of covalent intermediates in the biosynthesis of gramicidin S using high-resolution mass spectrometry.

For determination of multiple covalent intermediates bound to the ultra-large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry (MS) is a promising method to provide new mechanistic insight. Application of a quadrupole-Fourier-transform instrument (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestions of recombinant proteins afforded detection of two active site peptides (both ~13 kDa) that provided direct evidence for modules copurifying with their preferred amino acid substrates. Given the ability to detect multiple covalent intermediates in tandem, a competition experiment among several nonnatural substrates in parallel was performed using the first module. This defined mixture of acyl-enzyme intermediates was used to probe the selectivity of the condensation step producing a diversity of noncognate dipeptides on the second module.

Chemoenzymatic approaches for streamlined detection of active site modifications on thiotemplate assembly lines using mass spectrometry.

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

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.

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.

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.

Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity.

The lantibiotic lacticin 481 is synthesized on ribosomes as a prepeptide (LctA) and posttranslationally modified to its mature form. These modifications include dehydration of serines and threonines, followed by intramolecular addition of cysteines to the unsaturated amino acids, which generates cyclic thioethers. This process breaks eight chemical bonds and forms six newbonds and is catalyzed by one enzyme, LctM. We have characterized the in vitro activity of LctM, which completely processed a series of LctA mutants, displaying a permissive substrate specificity that holds promise for antibiotic engineering.