A method is described for the rapid identification of oligosaccharides employing a library of tandem MS spectra. Identification is aided by software that compares the sample tandem MS to those in the library. The method incorporates quadrupole time-of-flight mass spectrometry along with an annotated oligosaccharide (OS) structure library and the MassHunter Personal Compound Database and Library (PCDL) software. With an automated spectra search, OS structures in different samples are readily identified. This method is shown to be useful in the study of milk oligosaccharides but can be readily applied to oligosaccharide pools in other biological tissues.

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC-MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.

Abstract Introduction Tuberculous meningitis is a frequent extrapulmonary disease caused by Mycobacterium tuberculosis and is associated with high mortality rates and severe neurological sequelae. In an earlier study employing DNA microarrays, we had identified genes that were differentially expressed at the transcript level in human brain tissue from cases of tuberculous meningitis. In the current study, we used a quantitative proteomics approach to discover protein biomarkers for tuberculous meningitis. Methods To compare brain tissues from confirmed cased of tuberculous meningitis with uninfected brain tissue, we carried out quantitative protein expression profiling using iTRAQ labeling and LC-MS/MS analysis of SCX fractionated peptides on Agilent’s accurate mass QTOF mass spectrometer. Results and conclusions Through this approach, we identified both known and novel differentially regulated molecules. Those described previously included signal-regulatory protein alpha (SIRPA) and protein disulfide isomerase family A, member 6 (PDIA6), which have been shown to be overexpressed at the mRNA level in tuberculous meningitis. The novel overexpressed proteins identified in our study included amphiphysin (AMPH) and neurofascin (NFASC) while ferritin light chain (FTL) was found to be downregulated in TBM. We validated amphiphysin, neurofascin and ferritin light chain using immunohistochemistry which confirmed their differential expression in tuberculous meningitis. Overall, our data provides insights into the host response in tuberculous meningitis at the molecular level in addition to providing candidate diagnostic biomarkers for tuberculous meningitis.

Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, β-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, β-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.

Assays that are both specific and quantitative for target proteins are critical for preclinical validation of putative biomarkers. Such assays are typically multiplexed, multiple reaction monitoring (MRM) analyses which can provide the high-throughput required. Sensitivity is a key requirement for such assays as protein biomarker concentrations may be quite low in commonly used biofluids such as serum and plasma. Improving the sensitivity of LC/MS can be achieved by using nanoflow LC, and by enhancing the sampling and transmission of ions in the mass spectrometer. This study demonstrates the 5-10x sensitivity gain achieved for peptides using a triple quadrupole mass spectrometer modified with a dual ion funnel. The sensitivity achieved using a microfluidic-based nanoflow LC system compared to a standard LC system will be shown discussed.

RP-93 Comprehensive proteome analysis can be very challenging due to complexity and range of protein concentrations.Techniques such as 2D LC, pI-based fractionation and gel electrophoresis are typically employed to increase separation efficiency as a strategy for obtaining more peptide MS/MS spectra and thus increasing the number of proteins identified.Having a standard sample of moderate complexity facilitates comparison of different protein identification workflows.For this work, a complex proteomics standard consisting of a soluble extract from Pyrococcus furiosu was employed to evaluate the effectiveness of different fractionation schemes for increasing protein identification.The fractionation schemes evaluated included protein fractionation by 1D SDS-PAGE and RPLC as well as peptide fractionation by OFFGEL electrophoresis.A comparison of the proteins identified as well as the total number of proteins and peptides will be done for measuring the effectiveness and relative orthogonality of the different workflows.

RP-87 Analyzing intact proteins using quadrupole time-of-flight (Q-TOF) mass Spectrometry is very popular in the biopharma industry.Time-of-flight mass spectrometry offers excellent resolution and mass accuracy which are important for characterizing intact proteins.With the recent improvement of Q-TOF resolution from 20000 to 40000, we are able to fully resolve the isotope envelope of proteins up to 20kDa, which improves mass accuracy to 5-10ppm for these large molecules.The increased resolving power helped to resolve small post-translational modifications (PTM) and adducts of large molecules.In this study, intact proteins range from 5kDa - 150kDa were measured using Q-TOF with 20k and 40k resolution.Proteins up to 20 kDa were fully resolved at 40k resolution.Myoglobin sample measured with 40k resolution gave fully resolved isotope envelope.It was first deconvoluted using a Maximum Entropy charge deconvolution algorithm.In addition to the intact Myoglobin (16951 Da),Maximum Entropy also identified three oxidized Myoglobin molecules with maximum mass error of 23ppm.A different algorithm called molecular feature extraction (MFE) was also employed on this Myoglobin high resolution data.MFE used isotope cluster matching to calculate the mono isotopic mass.MFE also identified intact Myoglobin and its three oxidized forms with mass error less than 6ppm. In comparison, when the same Myoglobin is measured with lower than 20k resolution, the isotope envelope was not resolved. Only the intact Myoglobin can be correctly assigned with less than 50ppm mass accuracy.

In an effort to simplify and streamline compound identification from metabolomics data generated by liquid chromatography time-of-flight mass spectrometry, we have created software for constructing Personalized Metabolite Databases with content from over 15,000 compounds pulled from the public METLIN database (http://metlin.scripps.edu/). Moreover, we have added extra functionalities to the database that (a) permit the addition of user-defined retention times as an orthogonal searchable parameter to complement accurate mass data; and (b) allow interfacing to separate software, a Molecular Formula Generator (MFG), that facilitates reliable interpretation of any database matches from the accurate mass spectral data. To test the utility of this identification strategy, we added retention times to a subset of masses in this database, representing a mixture of 78 synthetic urine standards. The synthetic mixture was analyzed and screened against this METLIN urine database, resulting in 46 accurate mass and retention time matches. Human urine samples were subsequently analyzed under the same analytical conditions and screened against this database. A total of 1387 ions were detected in human urine; 16 of these ions matched both accurate mass and retention time parameters for the 78 urine standards in the database. Another 374 had only an accurate mass match to the database, with 163 of those masses also having the highest MFG score. Furthermore, MFG calculated a formula for a further 849 ions that had no match to the database. Taken together, these results suggest that the METLIN Personal Metabolite database and MFG software offer a robust strategy for confirming the formula of database matches. In the event of no database match, it also suggests possible formulas that may be helpful in interpreting the experimental results.

Reproducible and comprehensive sample extraction and detection of metabolites with a broad range of physico-chemical properties from biological matrices can be a highly challenging process. A single LC/MS separation method was developed for a 2.1 mm x 100 mm, 1.8 microm ZORBAX SB-Aq column that was used to separate human erythrocyte metabolites extracted under sample extraction solvent conditions where the pH was neutral or had been adjusted to either, pH 2, 6 or 9. Internal standards were included and evaluated for tracking sample extraction efficiency. Through the combination of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques in both positive (+) and negative (-) ion modes, a total of 2370 features (compounds and associated compound related components: isotopes, adducts and dimers) were detected across all pHs. Broader coverage of the detected metabolome was achieved by observing that (1) performing extractions at pH 2 and 9, leads to a combined 92% increase in detected features over pH 7 alone; and (2) including APCI in the analysis results in a 34% increase in detected features, across all pHs, than the total number detected by ESI. A significant dependency of extraction solvent pH on the recovery of heme and other compounds was observed in erythrocytes and underscores the need for a comprehensive sample extraction strategy and LC/MS analysis in metabolomics profiling experiments.

The characterization of a monoclonal antibody at low nanogram levels using an Agilent HPLC-Chip system coupled to an Agilent Accurate-Mass Q-TOF MS instrument is described. The superior sensitivity and mass accuracy of the HPLC-Chip LC/MS platform, combined with the powerful data processing capabilities of Agilent MassHunter and BioConfi rm software, enabled easy and rapid identifi cation of antibody heterogeneity and cleaved fragments. Robust, reliable, and easy to use, Agilent nanofl ow LC/MS technology is ideally suited for the routine analysis of biopharmaceuticals.