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Semi-automated liquid chromatography-mass spectrometric imaging platform for enhanced detection and improved data analysis of complex peptides.

ABSTRACT: A semi-automated analytical platform featuring the coupling of monolithic reversed-phase liquid chromatography (RPLC) to matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) has been developed and evaluated. This is the first time that LC separation is readily coupled to MS imaging detection for the analysis of complex peptide mixtures both qualitatively and quantitatively. Methacrylate-based monolithic column with C12 functional groups is fabricated for fast RPLC separation. The LC flow and matrix flow are collected on a commercially available MALDI plate which is mechanically controlled and analyzed with MALDI MSI subsequently. Both tryptic peptides digested from bovine serum albumin (BSA) and endogenous neuropeptides extracted from the blue crab Callinectes sapidus are analyzed with this novel LC-MSI platform. Compared with regular offline LC fractionation coupled with MALDI MS detection, LC-MSI exhibits significantly increased MS signal intensity due to retaining of temporal resolution from separation dimension via continuous sampling, which results in increased number of peptides detected and accurate quantitation. In addition, imaging signals enable improved data analysis based on either mass-to-charge ratio or retention time, which is extremely beneficial for the analysis of complex analytes. These findings have demonstrated the potential of employing LC-MSI platform for enhanced proteomics and peptidomics studies.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC3786203 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Semi-automated liquid chromatography-mass spectrometric imaging platform for enhanced detection and improved data analysis of complex peptides.

Zhang Zichuan Z Jiang Shan S Li Lingjun L

Journal of chromatography. A 20130326

A semi-automated analytical platform featuring the coupling of monolithic reversed-phase liquid chromatography (RPLC) to matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) has been developed and evaluated. This is the first time that LC separation is readily coupled to MS imaging detection for the analysis of complex peptide mixtures both qualitatively and quantitatively. Methacrylate-based monolithic column with C12 functional groups is fabricated for fast RPLC s ...[more]

PMID: 23623366

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Project description:RationaleLiquid chromatography/mass spectrometry (LC/MS) is currently considered to be a conventional glycomics analysis strategy due to the high sensitivity and ability to handle complex biological samples. Interpretation of LC/MS data is a major bottleneck in high-throughput glycomics LC/MS-based analysis. The complexity of LC/MS data associated with biological samples prompts the needs to develop computational tools capable of facilitating automated data annotation and quantitation.MethodsAn LC/MS-based automated data annotation and quantitation software, MultiGlycan-ESI, was developed and utilized for glycan quantitation. Data generated by the software from LC/MS analysis of permethylated N-glycans derived from fetuin were initially validated by manual integration to assess the performance of the software. The performance of MultiGlycan-ESI was then assessed for the quantitation of permethylated fetuin N-glycans analyzed at different concentrations or spiked with permethylated N-glycans derived from human blood serum.ResultsThe relative abundance differences between data generated by the software and those generated by manual integration were less than 5%, indicating the reliability of MultiGlycan-ESI in quantitation of permethylated glycans analyzed by LC/MS. Automated quantitation resulted in a linear relationship for all six N-glycans derived from 50 ng to 400 ng fetuin with correlation coefficients (R(2) ) greater than 0.93. Spiking of permethylated fetuin N-glycans at different concentrations in permethylated N-glycan samples derived from a 0.02 μL of HBS also exhibited linear agreement with R(2) values greater than 0.9.ConclusionsWith a variety of options, including mass accuracy, merged adducts, and filtering criteria, MultiGlycan-ESI allows automated annotation and quantitation of LC/ESI-MS N-glycan data. The software allows the reliable quantitation of glycan LC/MS data. The software is reliable for automated glycan quantitation, thus facilitating rapid and reliable high-throughput glycomics studies.

Project description:Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3-6 monosaccharides attached to specific serine residues can be extended by 20-200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-022-00092-3.

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Semi-automated liquid chromatography-mass spectrometric imaging platform for enhanced detection and improved data analysis of complex peptides.

Publications

Semi-automated liquid chromatography-mass spectrometric imaging platform for enhanced detection and improved data analysis of complex peptides.

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