Accurate mass analysis of phosphoramidites by electrospray mass spectrometry

A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.     

Protein mass analysis of histones

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.     

A mass spectrometric analysis of the water-splitting reaction

Earlier mass spectrometric measurements, in which oxygen evolution was measured following short saturating light flashes, indicated that with a time resolution of about 30 s no form of bound water and/or an oxidation product exists up to the redox state S₃ of the oxygen evolving center (R. Radmer and O. Ollinger, 1986, FEBS Lett 195: 285–289; K.P. Bader, P. Thibault and G.H. Schmid, 1987, Biochim Biophys Acta 893: 564–571). In the present study, isotope exchange experiments with H₂ ¹⁸O were performed under different experimental conditions. We found: a) the isotope exchange pattern is virtually the same at both pH 6.0 and 7.8, although marked structural changes of the PS II donor side are inferred to take place within this pH-range (Renger G., Messinger J. and Wacker U., 1992, Research in Photosynthesis, II: 329–332); b) injection of H₂ ¹⁸O at about 0°C gives rise to mass ratios of the evolved oxygen which markedly deviate from the theoretically expected values of complete isotope scrambling; and c) rapid injection of H₂ ¹⁸O into samples with high population of S₁ and S₂ and subsequent illumination with three and two flashes, respectively, spaced by a dark time of only 1.5 ms lead to similar ¹⁸O-labeling of the evolved oxygen. Based on the published data on the interaction with redox active amines, possible pathways of substrate exchange in the water oxidase are discussed.Abbreviations atom fraction of ¹⁸O - PS II Photosystem II - S_i redox states of the water oxidase - Y_z redox active tyrosine of polypeptide D1     

Numerical bias estimation for mass spectrometric mass isotopomer analysis

Mass spectrometric (MS) isotopomer analysis has become a standard tool for investigating biological systems using stable isotopes. In particular, metabolic flux analysis uses mass isotopomers of metabolic products typically formed from ¹³C-labeled substrates to quantitate intracellular pathway fluxes. In the current work, we describe a model-driven method of numerical bias estimation regarding MS isotopomer analysis. Correct bias estimation is crucial for measuring statistical qualities of measurements and obtaining reliable fluxes. The model we developed for bias estimation corrects a priori unknown systematic errors unique for each individual mass isotopomer peak. For validation, we carried out both computational simulations and experimental measurements. From stochastic simulations, it was observed that carbon mass isotopomer distributions and measurement noise can be determined much more precisely only if signals are corrected for possible systematic errors. By removing the estimated background signals, the residuals resulting from experimental measurement and model expectation became consistent with normality, experimental variability was reduced, and data consistency was improved. The method is useful for obtaining systematic error-free data from ¹³C tracer experiments and can also be extended to other stable isotopes. As a result, the reliability of metabolic fluxes that are typically computed from mass isotopomer measurements is increased.     

Feature extraction in the analysis of proteomic mass spectra

Feature extraction or biomarker selection is a critical step in disease diagnosis and knowledge discovery based on protein MS. Many studies have discussed the classification methods applied in proteomics; however, few could be found to address feature extraction in detail. In this paper, we developed a systematic approach for the extraction of mass spectrum peak apex and peak area with special emphasis on noise filtration and peak calibration. Application to a head and neck cancer data generated at the Eastern Virginia Medical School [Wadsworth, J. T., Somers, K. D., Cazares, L. H., Malik, G. et al.., Clin. Cancer Res. 2004, 10, 1625-1632] revealed that the new feature extraction method would yield consistent and highly discriminatory biomarkers.     

Imaging mass spectrometry statistical analysis

Imaging mass spectrometry is increasingly used to identify new candidate biomarkers. This clinical application of imaging mass spectrometry is highly multidisciplinary: expertise in mass spectrometry is necessary to acquire high quality data, histology is required to accurately label the origin of each pixel's mass spectrum, disease biology is necessary to understand the potential meaning of the imaging mass spectrometry results, and statistics to assess the confidence of any findings. Imaging mass spectrometry data analysis is further complicated because of the unique nature of the data (within the mass spectrometry field); several of the assumptions implicit in the analysis of LC-MS/profiling datasets are not applicable to imaging. The very large size of imaging datasets and the reporting of many data analysis routines, combined with inadequate training and accessible reviews, have exacerbated this problem. In this paper we provide an accessible review of the nature of imaging data and the different strategies by which the data may be analyzed. Particular attention is paid to the assumptions of the data analysis routines to ensure that the reader is apprised of their correct usage in imaging mass spectrometry research.     

离子阱串联质谱仪质荷比测量误差统计分析

离子阱串联质谱仪是蛋白质组研究中一种高通量,高灵敏度的分析仪器。目前对影响离子阱质谱仪质荷比测量误差的因素和数据产出后系统误差的校正方法还没有系统的分析。利用两批蛋白质标准品的数据和统计方法分析了离子阱质谱仪质荷比测量误差的分布规律,对测量的质荷比和信号强度对测量误差的影响进行了分析。在此基础上,提出了一种对质谱数据进行系统误差再校正的方法和一种根据信号强度确定误差容限的模型。     

Process analysis of the acetone-butanol fermentation by quadrupole mass spectrometry

Summary Mass spectrometric process analysis of the ABE fermentation can be performed. However, the constraints imposed by the conditions at the inlet system do not allow quantifications based on single compound calibrations. Calibrations can be performed with mixtures typical for the process.     

Posttranslational modifications of the lutropin receptor: mass spectrometric analysis

Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.     

Optimal analysis of complex protein mass spectra

Due to physical and chemical phenomena, a simple sample can give rise to a complex mass spectrum with many more peaks than the number of molecular species present in the sample. We link peaks within and between different spectra, and come up with an advanced analysis approach to produce reliable estimates of the molecule masses and abundances. By linking peaks, we can locate multiple‐charge peaks at the correct position in the spectrum, we can deconvolute complex regions with many overlapping peaks by including information from related regions with lower complexity and higher resolution, and we reduce the total number of observed peaks in a spectrum to a much smaller number of underlying molecular species. In this paper we properly model 29 952 peaks in 64 spectra, using only 39 location parameters and one shape parameter. This major reduction from many different molecules to a limited set of molecular species reduces the statistical test multiplicity for biomarker discovery and therefore we imply that the reduction should eventually increase the biomarker discovery power significantly, too.     

Spatially-correlated mass spectrometric analysis of microbe--mineral interactions

A new methodology for examining the interactions of microbes with heterogeneous minerals is presented. Imaging laser desorption Fourier transform mass spectrometry was used to examine the colonization patterns of Burkholderia vietnamiensis G4 (previously Burkholderia cepacia G4) on a heterogeneous basalt sample. Depth-profile imaging found that the bacterium preferentially colonized the plagioclase mineral phase within the basalt.     

Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharides

Oligosaccharides from hyaluronic acid and chondroitin 6-sulfate were prepared by digestion with testicular hyaluronidase and separated according to their degree of polymerization by gel-permeation chromatography. These materials were successively analyzed by negative-mode ion-spray mass spectrometry with an atmospheric-pressure ion source. An ion-spray interface was used to produce ions via the ion evaporation process, producing mass spectra containing a series of molecular species carrying multiple charges. Using two adjacent multiply charged molecular ions, the exact molecular weights up to the tetradecasaccharide were calculated with a precision of ±1 dalton. This type of mass spectrometry was also demonstrated to be feasible for the analysis of mixtures of oligosaccharides, including tetra-, hexa-, octa- and decasaccharides, from hyaluronic acid or chondroitin 6-sulfate without separation. Ion-spray mass spectrometry was thus shown to be applicable to the structural analysis of oligosaccharides from glycosaminoglycans.Abbreviations HA hyaluronic acid - Ch6S chondroitin 6-sulfate - GAG glycosaminoglycan - GlcA d-glucuronic acid - GlcNAc 2-acetamido-2-deoxy-d-glucose - GalNAc 2-acetamido-2-deoxy-d-galactose.     

糖链的生物质谱分析

糖链是重要的生物信息分子,在许多生理和病理过程中都发挥着独特作用。糖链结构非常复杂,具有微观不均一性,其分析和结构解析一直是糖生物学研究的瓶颈。质谱具有灵敏度高、可获得多种结构信息和适于分析混合物等优点,是糖链定性定量分析的一种理想手段。电喷雾电离质谱和基质辅助激光解析电离质谱两大生物质谱技术已被广泛应用于糖链的相对分子质量指纹谱分析、序列和连接方式测定及相对定量分析。对近年来以质谱为主要分析手段的糖链分析方法研究进展做一综述。     

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

ABSTRACT

The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.     

The use of mass spectrometry for the proteomic analysis of glycosylation

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.     

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.