Human tear fluid is a complex mixture containing high concentrations of proteins and is increasingly becoming an important source for studying protein biomarkers of eye-related diseases such as Graves' ophthalmopathy. Today, the Schirmer tear test is the most widely used technique for tear collection. However, sample handling and protein extraction from these strips have been highly challenging. Cutting and removal of the Schirmer strips after extraction, which may lead to sample loss prior to downstream analysis, are some of the challenges to consider. To address some of these limitations, we have developed a single-unit filter-aided method for both sample handling and protein extraction. In addition, we systematically investigated the most suitable conditions for protein extraction from these strips. Among the different extraction conditions applied, extraction with 100 mM ammonium bicarbonate containing 50 mM NaCl resulted in the highest number of identified proteins using one-dimensional liquid chromatography tandem mass spectrometry (LC-MS/MS). Moreover, 1526 proteins were identified when the optimized extraction method was combined with two-dimensional LC-MS/MS analysis, demonstrating the applicability of this novel approach to the study of the tear proteome. This dataset of identified proteins represents a comprehensive catalogue of the tear proteome and may serve as a list for future biomarker research.

Several peptide drugs are being manufactured illicitly, and in some cases they are being made available to the public before entering or completing clinical trials. At the request of Norwegian police and customs authorities, unknown pharmaceutical preparations suspected to contain peptide drugs are regularly subjected to analysis. In 2009, an unknown pharmaceutical preparation was submitted for analysis by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). The preparation was found to contain a 29 amino acid peptide with a C-terminal amide function. Based on the interpretation of mass spectrometric data, an amino acid sequence was proposed. The sequence is consistent with a peptide currently marketed under the name CJC-1295. CJC-1295 is a releasing factor for growth hormone and is therefore considered a Prohibited Substance under Section S2 of the WADA Prohibited List. This substance has potential performance-enhancing effects, it is readily available, and there is reason to believe that it is being used within the bodybuilding community.

No abstract

It remains to be elucidated whether some of these proteins can be used as markers for patients at risk for developing GO as well as useful indicators for disease activity.

No abstract

Diabetes is associated with increased risk of cardiovascular disease. Advanced glycation end-products (AGEs) are considered to be a major pathogenic factor for diabetic vascular complications. The levels of AGEs are increased in diabetic patients. We have studied the presence of the major AGE methylglyoxal (MGO)-derived hydroimidazolone in human aorta and carotid arteries, using immunohistochemistry (IHC), western blotting and mass spectrometry. By IHC, MGO-derived modifications were detected mainly associated with cells in intimal thickenings and cells in microvessels in adventitia. In type V lesions MGO-derived AGE was also present, extracellular in the necrotic core and in cells at the border of the core. The highest degree of modification was probably associated with cell nuclei. By western blotting and mass spectrometry fibrin(ogen), the cytoskeleton-associated protein moesin and the nuclear proteins lamin A and C were identified as putative main targets for MGO-derived modification. LC-MS/MS studies of fibrin(ogen) modified in vitro with low concentrations of MGO identified the sites that were most prone to modification. These results indicate that AGE modifications occur preferentially on specific proteins. The modification of these proteins may play a role in vascular dysfunction and development of atherosclerosis in diabetes.

No abstract

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometryIn the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.