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Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10??m OT columns suitable for small molecules, peptides and proteins.

ABSTRACT: We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10??m ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C₁₈-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today's capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2??g), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-?g amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is "fit-for-all", well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.

SUBMITTER: Vehus T

PROVIDER: S-EPMC5126632 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins.

Vehus T T Roberg-Larsen H H Waaler J J Aslaksen S S Krauss S S Wilson S R SR Lundanes E E

Scientific reports 20161129

We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C<sub>18</sub>-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today's capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved se ...[more]

PMID: 27897190

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Project description:Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.

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Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10??m OT columns suitable for small molecules, peptides and proteins.

Publications

Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins.

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