Phosphorylation is one of the most common of the reversible post-translational modifications, but also one of the most challenging. During the last few years, phosphopeptide analysis has benefited from the rapid improvement in biological mass spectrometry, but also from innovations such as immobilized metal affinity chromatography, the development of antibodies specific for phosphorylated amino acids, and most recently, chromatography using TiO2. During our analysis of the saliva phosphoproteome, we were concerned that there is a bias against monophosphorylated peptides when using TiO2 columns. We therefore decided to explore alternatives that rely on fractionation rather than affinity purification. Using the approach of Roepstorff and colleagues of packing columns in pipette tips to make disposable microcolumns, and combining strong cationic exchange (SCX) together with hydrophilic interaction liquid chromatography (HILIC), we have been able to fractionate peptides from digests of total saliva, and isolate phosphopeptides in a few fractions with a minimum of non-phosphorylated peptides. In preliminary experiments using about 6 μg of saliva, we have been able to map out phosphorylation sites from proteins such as the salivary acid proline-rich phosphoprotein 1/2, the salivary α-amylase, and statherin. A comparison of isolation of phosphopeptides using TiO2 vs. a combination of SCX and HILIC will be presented.

Introduction. The Bronchopulmonary Dysplasia (BPD) or Mikity Wilson Syndrome (MWS) are chronic lung diseases with several pathogenic mechanisms. They can be associated with atypical infections by U. urealyticum, C. trachomatis, and Mycoplasma. Objective. We researched pulmonary infections in 10 VLBWN with BPD or MWS and had negative cell cultures by PCR in tracheobronchial samples. Methods. We studied VLBWN in our institution with BPD (n 6) or WMS (n 4) (with oxygen dependence and images lung radiographic of lung damage with fibrosis and cystic areas) as diagnosis and negative cell culture to atypical pathogens. We took new tracheobronchial samples at 28 days of life and send them Virology Department to PCR and infection determination. First. Tracheobronchial aspirate was took with saline solution 0.9% and stored in aliquots at 20°C until needed. Second. DNA isolation by proteinase K method and we used gel electrophoresis and densitometer to determine DNA concentration. Third. Determinate polymerase chain reaction (PCR). DNA concentration and extraction process were included as samples to identify background bacterial sequences present in reagents and supplies. Results. We found 7/10 positive infections, 3/6 C. trachomatis infections in neonates with BPD and 4/4 with MWS: U. urealyticum (1), Mycoplasma genitalium (1), C. trachomatis (1) and M. pneumoniae (1). Conclusions. We should researched atypical pulmonary infections by intracellular pathogens by another methods with more sensitivity as PCR to explain inflammatory response and lung damage. 1125 Comparative Proteomic Analyses of Normal and Normal Pressure Hydrocephalus in Human Cerebrospinal Fluid