Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O-GlcNAc)-modified counterparts. Solid-phase enzymatic dephosphorylation proved to be a viable tool to condition O-GlcNAcylated peptide in mixtures with phosphopeptides for selective affinity purification. Acetylation, as an integral step of the sample-preparation method, precluded reduction in recovery of the thiolation substrate caused by intrapeptide lysine-dehydroalanine cross-link formation. The solid-phase analytical platform provides robustness and simplicity of operation using equipment readily available in most biological laboratories and is expected to accommodate additional chemistries to expand the scope of solid-phase serial derivatization for protein structural characterization.     

N-Terminal Protein Characterization by Mass Spectrometry after Cyanogen Bromide Cleavage using Combined Microscale Liquid- and Solid-Phase Derivatization

A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTip_C18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate. In the subsequent steps, the peptides were exposed in situ to hydroxylamine for reversal of potential hydroxyl group acylation, followed by reductive release of the disulfide-linked biotinamido moiety from the derivatives. The selectively thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol-sepharose, leaving the N-terminal fragment in the flow-through fraction. The use of the reversed-phase support as a venue for postcleavage serial modification proved instrumental to ensure throughput and completeness of derivatization. By this sequence of solid-phase reactions, the N-terminal peptide could be recognized uniquely in the MALDI-mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-picomole amounts of model protein. The N-terminal CNBr fragments were retrieved selectively from the affinity support. The sample preparation method provides for robustness and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to gel-separated proteins.     

Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.     

Host-Polarized Cell Growth in Animal Symbionts

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How fast is fast? Eco‐evolutionary dynamics and rates of change in populations and phenotypes

It is increasingly recognized that evolution may occur in ecological time. It is not clear, however, how fast evolution – or phenotypic change more generally – may be in comparison with the associated ecology, or whether systems with fast ecological dynamics generally have relatively fast rates of phenotypic change. We developed a new dataset on standardized rates of change in population size and phenotypic traits for a wide range of species and taxonomic groups. We show that rates of change in phenotypes are generally no more than 2/3, and on average about 1/4, the concurrent rates of change in population size. There was no relationship between rates of population change and rates of phenotypic change across systems. We also found that the variance of both phenotypic and ecological rates increased with the mean across studies following a power law with an exponent of two, while temporal variation in phenotypic rates was lower than in ecological rates. Our results are consistent with the view that ecology and evolution may occur at similar time scales, but clarify that only rarely do populations change as fast in traits as they do in abundance.     

Identification and characterization of DUSP27, a novel dual-specific protein phosphatase

A novel human dual-specific protein phosphatase (DSP), designated DUSP27, is here described. The DUSP27 gene contains three exons, rather than the predicted 4-14 exons, and encodes a 220 amino acid protein. DUSP27 is structurally similar to other small DSPs, like VHR and DUSP13. The location of DUSP27 on chromosome 10q22, 50 kb upstream of DUSP13, suggests that these two genes arose by gene duplication. DUSP27 is an active enzyme, and its kinetic parameters and were determined. DUSP27 is a cytosolic enzyme, expressed in skeletal muscle, liver and adipose tissue, suggesting its possible role in energy metabolism.     

Rapid and sustained improvements in health-related quality of life,fatigue, and other patient-reported outcomes in rheumatoid arthritis patients treated with certolizumab pegol plus methotrexate over 1 year: results from the RAPID 1 randomized controlled trial

Introduction

The objective of this study was to assess the impact of certolizumab pegol (CZP) treatment on health-related quality of life (HRQoL), fatigue and other patient-reported outcomes (PROs) in patients with rheumatoid arthritis (RA).

Methods

Patients with active RA (N = 982) were randomized 2:2:1 to subcutaneous CZP (400 mg at weeks 0, 2 and 4; followed by CZP 200 mg or 400 mg) plus methotrexate (MTX) every other week, or placebo (PBO) plus MTX. PRO assessments included HRQoL, fatigue, physical function, arthritis pain and disease activity. Adjusted mean changes from baseline in all PROs were obtained using analysis of covariance (ANCOVA) applying last observation carried forward (LOCF) imputation. The proportion of patients achieving clinically meaningful improvements in each PRO was obtained using logistic regression and by applying non-responder imputation to missing values after rescue medication or withdrawal. The correlations between PRO responses and clinical responses were also assessed by tetrachoric correlation using non-responder imputation.

Results

Patients treated with CZP plus MTX reported significant (P < 0.001), clinically meaningful improvements in HRQoL at the first assessment (week 12); reductions in fatigue, disease activity and pain and improvements in physical function were reported at week 1. In particular, CZP-treated patients reported improvements in mental health. Mean changes from baseline in the SF-36 Mental Component Summary (MCS) at week 52 for CZP 200 mg and 400 mg plus MTX, and PBO plus MTX were 6.4, 6.4 and 2.1, respectively (P < 0.001). In addition, mental health and vitality scores in CZP-treated patients approached age- and gender-adjusted US population norms. Improvements in all PROs were sustained. Similar benefits were reported with both CZP doses. Changes in SF-36 MCS scores had the lowest correlation with disease activity scores (DAS28) and American College of Rheumatology 20% improvement (ACR20) response rates, while improvements in pain showed the highest correlation.

Conclusions

Treatment with CZP plus MTX resulted in rapid and sustained improvements in all PROs, indicating that the benefits of CZP extend beyond clinical efficacy endpoints into areas that are more relevant and meaningful for patients on a daily basis.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00152386","term_id":"NCT00152386"}}NCT00152386.     

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque,a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.¹Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.⁴ While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.² The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR ''window'' can substantially improve the potential for in vivo imaging.^2,5Inflammatory cysteine proteases have been well studied using activatable NIRF probes¹⁰, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis⁸. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.^3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology⁶, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.¹⁰ Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque. In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,⁶ is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.^11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.Download video file.^{(61M, mov)}