We have identified over 100 protein and peptide components of normal human seminal fluid.

More than 10 million people are thought to be infected with Trypanosoma cruzi, primarily in the Americas. The clinical manifestations of Chagas' disease (CD) are variable, but most subjects remain asymptomatic for decades. Only 15 to 30% eventually develop terminal complications. All current diagnostic tests have limitations. New approaches are needed for blood bank screening as well as for improved diagnosis and prognosis. Sera from subjects with asymptomatic CD (n ‫؍‬ 131) were compared to those from uninfected controls (n ‫؍‬ 164) and subjects with other parasitic diseases (n ‫؍‬ 140), using protein array mass spectrometry. To identify biomarkers associated with CD, sera were fractionated by anion-exchange chromatography and bound to two commercial ProteinChip array chemistries: WCX2 and IMAC3. Multiple candidate biomarkers were found in CD sera (3 to 75.4 kDa). Algorithms employing 3 to 5 of these biomarkers achieved up to 100% sensitivity and 98% specificity for CD. The biomarkers most useful for diagnosis were identified and validated. These included MIP1 alpha, C3a anaphylatoxin, and unusually truncated forms of fibronectin, apolipoprotein A1 (ApoA1), and C3. An antipeptide antiserum against the 28.9-kDa C terminus of the fibronectin fragment achieved good specificity (90%) for CD in a Western blot format. We identified full-length ApoA1 (28.1 kDa), the major structural and functional protein component of high-density lipoprotein (HDL), as an important negative biomarker for CD, and relatively little full-length ApoA1 was detected in CD sera. This work provides proof of principle that both platform-dependent (i.e., mass spectrometry-based) and platform-independent (i.e., Western blot) tests can be generated using high-throughput mass profiling.

A range of low molecular weight compounds (<800 Da) have been examined by matrix-assisted laser desorption time-of-flight mass spectrometry to demonstrate the general analytical utility of this technique. The compound classes investigated included: carbohydrates, quaternary ammonium salts, sterols, nucleosides, purine and pyrimidine bases, amino acids, catecholamines, opioids, antibiotics, prostaglandins and a range of macrocyclic metal complexes of porphyrins and phthalocyanines. Many of the compounds tested are of biochemical, geochemical, industrial and/or therapeutic interest. Most organic analytes gave intense protonated molecules, but some were characterized by sodium adduct ions (i.e., [M + Na]'). The metal-containing compounds formed radical-cation molecular ions. In all instances the ions detected could be assigned, and excellent agreements between calculated and experimental mass values were obtained.Matrix-assisted laser desorption/ionization (MALDI) combined with time-of-flight (TOF) mass spectrometry has found general acclaim as an approach to syntheticand bio-polymer analysis. MALDI allows the direct analysis of mixtures, and hence, analyte impurities can usually be identified and characterized. Sub-picomole amounts of sample are adequate for multiple analyses, results are available in minutes, and most of the sample can be recovered directly from the target. The increasing availability of inexpensive commercial instruments has promoted widespread use of MALDI-TOFMS, particularly for biopolymers, but studies of compounds of low molecular weight (i.e., ca. 800 Da or less) have been re~tricted.'-~ Here we present experimental results for the MALDI analysis of a range of low molecular weight compounds including carbohydrates, quaternary ammonium salts, sterols, nucleosides, purine and pyrimidine bases, amino acids, catecholamines, opioids, antibiotics, prostaglandins and a range of macrocyclic metal complexes of porphyrins and phthalocyanines. Many of the compounds tested are of biochemical, geochemical, industrial and/or therapeutic significance. EXPERIMENTAL InstrumentationAnalyses were performed on a Finnigan Lasermat MALDI linear time-of-flight mass spectrometer (Finnigan MAT, Heme1 Hempstead, UK). Much of the work described in this paper was performed on a standard Lasermat, but later data were obtained on the instrument following upgrade to Lasermat 2000 specifications. The system uses a nitrogen laser (337 nm; 3 ns pulse duration), and the laser fluence is adjusted by a variable-beam attenuator. Samples were applied to stainless-steel slides with a circular sample application area of 2 mm diameter. The Lasermat software allows the user to irradiate one of four possible target regions * Author for correspondence within this area. Under standard operating conditions, and for the mass range 100-1000Da, the resolving power ( m / A m , full width at half maximum height) of the Lasermat 2000 is in the range ca. 100-200 (or 50-100 for the Lasermat). Materials and methodsWater was purified by a Millipor...

To unlock the full potential of disease modifying treatments, it is essential to develop early biomarkers for Alzheimer's disease (AD). For practical reasons, blood-based markers that could provide a signal at the stage of mild cognitive impairment (MCI) or even earlier would be ideal. Using the proteomic approach of isobaric tagging for relative and absolute quantitation (iTRAQ), we compared the plasma protein profiles of MCI, AD, and cognitively normal control subjects from two independent cohorts: the Sydney Memory and Ageing Study (261 MCI subjects, 24 AD subjects, 411 controls) and the Hunter Community Study (180 MCI subjects, 153 controls). The objective was to identify any proteins that are differentially abundant in MCI and AD plasma in both cohorts, since they might be of interest as potential biomarkers, or could help direct future mechanistic studies. Proteins representative of biological processes relevant to AD pathology, such as the complement system, the coagulation cascade, lipid metabolism, and metal and vitamin D and E transport, were found to differ in abundance in MCI. In particular, levels of complement regulators C1 inhibitor and factor H, fibronectin, ceruloplasmin, and vitamin D-binding protein were significantly decreased in MCI participants from both cohorts. Several apolipoproteins, including apolipoprotein AIV, B-100, and H were also significantly decreased in MCI. Most of these proteins have previously been reported as potential biomarkers for AD; however, we show for the first time that a significant decrease in plasma levels of two potential biomarkers (fibronectin and C1 inhibitor) is evident at the MCI stage.

The olefins Ph,P(X)CH=CHR [X=lone pair, 0, S, CH31; R=CH3, Ph, P(X)PhJ have been prepared and their 'H, 13C and "P N M R spectra measured. trans 'qP(IV)C] (range 18.3-25.7HzI is greater than cis 3J[P(IV)C] (range 6.9-11 Hz) but this relationship does not hold for P(II1) compounds. In the "P spectra the E isomer absorbs to higher field than the Z isomer for P(II1) and P(IV) compounds. The 'H data are in accord with previous results; average substituent shielding coefficients for Ph,P(X) substituted alkenes are reported.Numerous studies of the 'H, 13C and 31P spectra of various classes of organophosphorus compounds have been published. Both ,J(PH) and ,J(PC) appear to follow a Karplus-type relationship,lZ2 though recent results have shown some unexpected variations when the phosphorus is trivalent.2h However, there are very few data in the literature on the differences expected in the 13C and 31P spectra of EZ alkene pairs. Such information is desirable for structure elucidation and for determining the steric course of reactions. In addition, since replacement of vinylic halogen by phosphorus is chemically simple, and reported to proceed stereo~pecifically,~ the introduction of a spin-active substituent could greatly simplify the analysis of I3C and/or 'H spectra of vinylic halides.We describe here the results of a 'H, 13C and 31P study of eight pairs of geometrical isomers: the phosphines (A; R = CH, or Ph) and their corresponding oxides, sulphides and methiodides. The 31P data for some symmetrical diphosphorus substituted ethenes are also included. Ph,PCH=CHR A EXPERIMENTALThe propenyl and styryl phosphines were prepared by reaction of diphenylphosphinous chloride with the Grignard reagent obtained from the appropriate halide. Commercially available 2-bromostyrene was predominantly the E isomer and a sample of the Z isomer was prepared by the method of Cristol and N o r r i~.~ 1-Bromopropene was largely (c. go0/,) the Z isomer. The stereochemistry of the halides was determined by 'H and 13C NMR and confirmed for the phosphines by isolating the major component of each mixture as its sulphide and oxide and comparing their melting points with literature values. The styrylphosphine 5 Z was isolated as a crystalline solid, m.p. 93-95'. The preparation of the phosphines was * Author to whom correspondence should be addressed. stereoselective though some inversion at vinylic carbon occurred, probably at the Grignard preparation stage. Stereomutation was most pronounced for the 2-styryl compound. Measurements were made only on crystalline 2 isomer obtained from this mixture.The diphosphines were prepared from the corresponding dihalide and lithium diphenylphosphide in tetrahydr~furan.~ The disulphides and dioxides are known compounds and were prepared by conventional methods.3-Methyl-1-0x0-1-phenylphosphol-2-ene (19) was a commercial sample.C and 31P spectra were determined on a Bruker WP-60-FT spectrometer at 15.08 and 24.28 MHz, respectively, using a sweep width of 3750 Hz and 4 K real data points. Carbons without hydr...

Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer-generated rankings. Results were verified using N-terminal protein microsequencing. The combined use of amino acid composition and MALDI-TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4-10 tryptic digest fragments). A further two parameters, estimated M(r) and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.

Background. Management of pediatric pulmonary hypertension (PH) remains challenging. We have assessed a panel of circulating proteins in children with PH to investigate their value as predictive and/or prognostic biomarkers. From these determinations, we aim to develop a practical, noninvasive tool to aid in the management of pediatric PH. Methods. Twelve cytokines and growth factors putatively associated with lung or vascular disease were examined in plasma specimens from 70 children with PH using multiplex protein array technology. Associations between hemodynamics, adverse events, and protein markers were evaluated. Results. Epidermal growth factor (EGF) and IL-6 were associated with important hemodynamics. Of the twelve proteins, VEGF and IL-6 were significantly, univariately associated with the occurrence of an adverse event, with odds ratios (95% confidence intervals) of 0.56 (0.33–0.98) and 1.69 (1.03–2.77), respectively. When hemodynamic predictors were combined with protein markers, the ability to predict adverse outcomes within the following year significantly increased. Conclusions. Specific circulating proteins are associated with hemodynamic variables in pediatric PH. If confirmed in additional cohorts, measurement of these proteins could aid patient care and design of clinical trials by identifying patients at risk for adverse events. These findings also further support a role for inflammation in pediatric PH.

No abstract