Claes Lindberg

Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

Transforming growth factor-β1 (TGF-β1) induces α-smooth muscle actin (α-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased α-SMA expression. Because TGF-β1 is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-β1 alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [35S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed ∼2500 proteins in the pIinterval of 3–10. Treatment of TGF-β1 led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-β1, and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-β1 induces not only α-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of α-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.

Quantitative high-performance liquid chromatography-tandem mass spectrometry method for the analysis of free desmosines in plasma and urine.

A rapid method for the determination of the sum of free desmosine and isodesmosine in human plasma and urine is described. Efficient sample clean-up prior to LC-MS/MS analysis is mandatory for detection of free desmosines in plasma samples. The combination of ultra-filtration and a two-step solid phase extraction minimizes the sample complexity and ion suppression effects. The flow through from the ultra filtration is passed through a C18 resin and then the target analytes are trapped and enriched on a mixed mode solid phase extraction material. The combination of these three orthogonal sample preparation steps allows detection of endogenous free desmosines in plasma from healthy individuals. An analytical column packed with porous graphitic carbon material enables the retention of the polar desmosine analytes, which are measured by electrospray ionization tandem mass spectrometry. Deuterium labeled isodesmosine is added as internal standard and a linear calibration curve was constructed in the range of 0.1-2.0 nmol/L for plasma samples and 5-200 nmol/L for urine samples. These results demonstrate that the described LC-MS/MS method provides sensitive, repeatable and accurate quantification of free desmosines in plasma and urine samples.

Blood biomarkers and measures of pulmonary function-A study from the Swedish twin registry.

OBJECTIVE There is great need of biomarkers for research and clinical purposes in COPD. This study explored the relationships between ten putative plasma biomarkers of COPD and physiological measures of reduced lung function. METHODS FEV(1), FVC, residual volume/total lung capacity (RV/TLC) and CO diffusion capacity (D(L)CO) were assessed in 357 subjects from the Swedish Twin Registry. The lung function measures were studied in relation to plasma levels of desmosines, C-reactive protein (CRP), plasminogen inhibitor activator (PAI-1) concentration and activity, tissue inhibitor of metalloproteinase (TIMP-1), clara cell protein 16 (CC16), surfactant protein D (SPD), matrix metalloproteinase 9 (MMP-9), hepatocyte growth factor (HGF) and interleukin (IL)-8. RESULTS After adjustments for age, sex, height, BMI and smoking, FEV(1) was significantly associated with PAI-1 activity and desmosines. RV/TLC was significantly associated with CC16, PAI-1 concentration and PAI-1 activity, and D(L)CO was significantly associated with desmosines, TIMP-1 and CRP. When the multivariate analysis was restricted to subjects with COPD (i.e., FEV(1)/FVC < 0.70), CRP and desmosines were inversely associated with lung function. CONCLUSION Several biomarkers were associated with lung function in this cross-sectional study. Especially CRP and desmosines could be useful markers to assess disease severity in subjects with COPD.

Profiling of endogenous peptides by multidimensional liquid chromatography: On-line automated sample cleanup for biomarker discovery in human urine

A simple and flexible system, employing a column switching technique, has been designed to allow the analysis of peptides and proteins smaller than 15 kDa by molecular weight in filtered urine samples by performing a direct on-column injection utilising simultaneous sample clean-up and trace enrichment. The positively charged peptides and small proteins in the sample are attracted to the inner, negatively charged pore structure of the RAM-SCX column while the larger proteins and uncharged or negatively charged compounds are excluded. After preconditioning with the biological sample, large amounts of sample can be injected. Several important and adjustable parameters for the proper use of a RAM-SCX column are described and discussed. The main parameters being: i) the column is sensitive to sample overloading, which may result in drastic changes in the adsorption of peptides; ii) adsorption appears to be flow-rate and concentration dependent, as the sample molecules need time to penetrate into the internal pore structure in order to find complimentary orientated adsorption sites; iii) dilution and pH adjustment of sample during the loading process. The biocompatibility and proof-of-principle of this separation platform was demonstrated using human urine samples. Data are presented on repeatability as well as on the reproducibility of different synthesised batches of restricted access material (RAM).

Liquid chromatography–tandem mass spectrometry approach for quantification of mucins from sputum using 13C,15N-labeled peptides as internal standards

Mucins are of great interest owing to their involvement in physiological and pathological processes in the airways. A method that allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic selected reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction, and alkylation prior to tryptic digestion. Solid phase extraction using C18 sorbent was used for sample cleanup prior to the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A cysteine-containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards, and linear calibration curves were constructed in the range of 0.3 to 40 pmol/L. Both mucins could be determined with a precision of 6 to 19% and an accuracy of 98 to 114%. The method is transferable to robotics and is suitable to be run in a 96-well format.