Serum biomarker discovery in renal cancer using 2-DE and prefractionation by immunodepletion and isoelectric focusing; increasing coverage or more of the same?

As an initial screen for novel markers of renal cancer and to minimise background heterogeneity, we have compared the within-patient profiles of serum samples from seven patients pre- and post-nephrectomy. Samples were depleted of six of the most abundant proteins using Agilent's multiple affinity removal system (MARS) followed by solution-phase IEF prior to separation by 2-DE using narrow range IPG Strips, with a total of 84 gels. The reproducibility of the various steps was demonstrated and an approximate two-fold increase (from 374 to 779) in the number of protein spots observed in the pH region 4.

Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle.

The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (beta) of 120-125 degrees with the filament axis.

Impact of preanalytical variables on the analysis of biological fluids in proteomic studies.

Although proteomic technologies have been enthusiastically embraced in the field of biomarker discovery and particularly with biological fluids, it is increasingly being recognized that pre-analytical effects, i.e. those occurring prior to the point of actual sample analysis and including factors such as sample processing and storage, can exert marked influences on the results obtained.

Resistance to the tubulin-binding agents in renal cell carcinoma: no mutations in the class I beta-tubulin gene but changes in tubulin isotype protein expression.

Purpose: The primary purpose of this study was to determine whether mutations of the class I beta-tubulin gene may be implicated in the inherent resistance to tubulin-binding agents (TBA) in renal cancer, with a small number of samples and cell lines also being examined for class I and III beta-tubulin isotype protein expression.

Experimental Design: DNA was extracted from 90 renal tumors and the class I beta-tubulin gene analyzed for mutations. For each sample, eight PCRs were used to cover the complete coding sequence with intronic primers ensuring highly homologous pseudogenes were not coamplified.

Intrinsic chemotherapy resistance to the tubulin-binding antimitotic agents in renal cell carcinoma.

Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins. The IC(50) values for vincristine correlated positively with PgP expression (r = 0.

Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies.

A primary objective of many protein expression studies is to define expression patterns that can distinguish between normal and diseased states, enabling a better understanding of molecular events associated with disease development and progression and ultimately potentially finding novel markers or therapeutic targets. Exploration and confirmation of many proteins is often done using Western blotting with normalization against "housekeeping proteins", such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, or beta-tubulin, to correct for protein loading and factors, such as transfer efficiency. Increasingly, in studies examining gene transcript levels, it has been shown that some of the commonly used housekeeping genes may be unsuitable due to the influence of various physiological and pathological factors on their expression.

Bifunctional rhodamine probes of Myosin regulatory light chain orientation in relaxed skeletal muscle fibers.

The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30 degrees C; approximately 60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution-the maximum entropy distribution-consistent with the polarized fluorescence data.

In situ orientations of protein domains: troponin C in skeletal muscle fibers.

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament.

NMR structure of a bifunctional rhodamine labeled N-domain of troponin C complexed with the regulatory "switch" peptide from troponin I: implications for in situ fluorescence studies in muscle fibers.

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al.

Orientation changes of the myosin light chain domain during filament sliding in active and rigor muscle.

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor.