Methods for treating MS/MS data to achieve accurate peptide identification are currently the subject of much research activity. In this study we describe a new method for filtering MS/MS data and refining precursor masses that provides highly accurate analyses of massive sets of proteomics data. This method, coined "postexperiment monoisotopic mass filtering and refinement" (PE-MMR), consists of several data processing steps: 1) generation of lists of all monoisotopic masses observed in a whole LC/MS experiment, 2) clusterization of monoisotopic masses of a peptide into unique mass classes ( MS has evolved into a robust and powerful tool for analyzing biomolecules. For example, MS is presently a preferred analytical tool for proteomics experiments in which dynamic populations of proteins are to be identified and quantified. In shotgun proteomics, hundreds or thousands of proteins are routinely identified through LC/MS/MS experiments using the proteolytic digests of proteome samples. During these LC/ MS/MS experiments, peptides are dynamically selected for fragmentation; the resultant tandem mass spectra (usually on the order of 10 5 spectra) are subsequently subjected to database searching (e.g. SEQUEST or Mascot) to best describe or annotate the peptide.Searching a large set of tandem mass spectra against a protein sequence database is a computationally expensive process; the resultant peptide spectral matches (PSMs) 1 are prone to false-positive (FP) identifications, which have led to the development of several validation methods (1, 2). Acquisition of high quality tandem mass spectra is critical to the whole process of protein identification through the shotgun approach because the measured fragment ion masses are compared with the theoretical values predicted by the search algorithms; any factors that adversely affect the acquisition of tandem mass spectra would undoubtedly lead to incorrect peptide assignments. Venable and Yates (3) systematically studied the effects that variability in tandem mass spectral quality had on peptide identification (i.e. peptide scores) and suggested an approach involving both presearch spectral averaging and postsearch XCorr averaging.Large portions (up to 90%) of tandem mass spectral data are not assigned to peptide sequences (4, 5) for many reasons, such as the presence of unknown protein sequences, the possibility of chemical (or post-translational) modification having occurred, insufficient spectral quality for high confi-

Plasma membrane calcium ATPases PMCA1 and PMCA4 regulate osteoclast differentiation and survival by regulating NFATc1 and NO.

aPrediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to NACT) to discover serum biomarkers of chemoresistance using a label-free liquid chromatography-tandem MS method. By performing a series of statistical analyses of the proteomic data, we selected thirteen biomarker candidates and tested their differential serum levels by Western blotting in 13 independent samples (eight patients sensitive to and five patients resistant to NACT). Among the candidates, we then selected the final set of six potential serum biomarkers (AHSG, APOB, C3, C9, CP, and ORM1) whose differential expression was confirmed in the independent samples. Finally, we demonstrated that a multivariate classification model using the six proteins could predict responses to NACT and further predict relapse-free survival of patients. In summary, global N-glycoproteome profile in serum revealed a protein pattern predictive of the responses to NACT, which can be further validated in large clinical studies. Molecular & Cellular Proteomics

Accurate assignment of monoisotopic precursor masses to tandem mass spectrometric (MS/MS) data is a fundamental and critically important step for successful peptide identifications in mass spectrometry based proteomics. Here we describe an integrated approach that combines three previously reported methods of treating MS/MS data for precursor mass refinement. This combined method, "integrated Post-Experiment Monoisotopic Mass Refinement" (iPE-MMR), integrates steps: 1) generation of refined MS/MS data by DeconMSn; 2) additional refinement of the resultant MS/MS data by a modified version of PE-MMR; 3) elimination of systematic errors of precursor masses using DtaRefinery. iPE-MMR is the first method that utilizes all MS information from multiple MS scans of a precursor ion including multiple charge states, in an MS scan, to determine precursor mass. By combining these methods, iPE-MMR increases sensitivity in peptide identification and provides increased accuracy when applied to complex highthroughput proteomics data.

a b s t r a c tOsteoclasts differentiate from macrophage-lineage cells to become specialized for bone resorption function. By a proteomics approach, we found that Lyn was down-regulated by the osteoclast differentiation factor, receptor activator of NF-jB ligand (RANKL). The forced reduction of Lyn caused a striking increase in the RANKL-induced PLCc1, Ca 2+ , and NFATc1 responses during differentiation. These data suggest that Lyn plays a negative role in osteoclastogenesis by interfering with the PLCc1-mediated Ca 2+ signaling that leads to NFATc1 activation. Consistent with the in vitro results, in vivo injection of Lyn specific siRNA into mice calvariae provoked a fulminant bone resorption. Our study provides the first evidence of the involvement of Lyn in the negative regulation of osteoclastogenesis by RANKL.

Capillary RPLC/ESI-MS (cRPLC/ESI-MS) is one of the most powerful analytical tools for current proteomic research. The development of cRPLC techniques coupled online to a mass spectrometer has focused on increasing the separation efficiency, detection sensitivity, and throughput. Recently, the use of high-pressure (over 10,000 psi) LC systems that utilize long, small inner diameter capillary columns has gained much attention for proteomic analyses. In this study, we developed an ultrahigh-pressure dual online SPE/capillary RPLC (DO-SPE/cRPLC) system. This LC system employs two online SPE columns and two capillary columns (75 microm inner diameter x 1 m length) in a single separation system, and has a maximum operating pressure of 10,000 psi. This DO-SPE/cRPLC system is capable of providing high-resolution separation in addition to several other advantageous features, such as high reproducibility in terms of the LC retention time, rapid sample injection, online desalting, online sample enrichment of dilute samples, and increased throughput as a result of essentially removing the column equilibration time between successive experiments. We coupled the DO-SPE/cRPLC system online to a tandem mass spectrometer to allow high-throughput proteomic analyses. In this paper, we demonstrate the efficiency of this DO-SPE/cRPLC/MS/MS system by its use in the analyses of proteomic samples exhibiting different levels of complexity.

The microcapillary liquid chromatography (µLC)/tandem mass spectrometry (MS/MS) system has become a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteome-wide identification of peptides and proteins rely heavily on producing an accurate and reproducible solvent-composition gradient throughout microcapillary separation columns to improve LC separation. With the recent advent of targeted proteomic approaches utilizing the LC retention time as a physicochemical parameter for peptides, high reproducibility of LC separation additionally becomes an important factor. In this study, column temperature elevation is utilized to improve reproducibility and separation efficiency of the µLC-MS/MS system. The simple incorporation of a semi-rigid gas line heater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up to 60 °C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlled temperature elevation on the peptide separation efficiency and reproducibility. In addition to the increased reproducibility in peptide elution time due to the controlled column temperature, the temperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowed a higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements in the performance of µLC systems.

A multi-functional liquid chromatography system that performs 1-dimensional, 2-dimensional (strong cation exchange/reverse phase liquid chromatography, or SCX/RPLC) separations and online phosphopeptide enrichment using a single binary nano-flow pump has been developed. With a simple operation of a function selection valve equipped with a SCX column and a TiO2 (titanium dioxide) column, a fully automated selection of three different experiment modes was achieved. Because the current system uses essentially the same solvent flow paths, the same trap column, and the same separation column for reverse-phase separation of 1D, 2D, and online phosphopeptides enrichment experiments, the elution time information obtained from these experiments is in excellent agreement, which facilitates correlating peptide information from different experiments. The final reverse-phase separation of the three experiments is completely decoupled from all of function selection processes; thereby salts or acids from SCX or TiO2 column do not affect the efficiency of the reverse-phase separation.