A chip‐based amide‐HILIC LC/MS platform for glycosaminoglycan glycomics profiling

Abstract

A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple mass spectrometry with an on-line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide-silica hydrophilic interaction chromatography (HILIC) in a chip-based format for LC/MS of heparin, heparan sulfate and chondroitin/dermatan sulfate GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built-in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate groups and acidic sulfate groups. In summary, chip based amide-HILIC LC/MS is an enabling technology for GAG glycomics profiling.

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

“…Samples were resuspended in 30 ml ultrapure ddH 2 O and 0.1% formic acid for identification by ion trap LC-MS analysis. Separation of peptides was performed with a nanoflow Agilent 1200 series system, equipped with a Zorbax 300SB C18 5 mm, 4 mm 40 nl pre-column and a Zorbax 300SB C18 5 mm, 43 mm  75 mm analytical reversedphase column using the HPLC-Chip technology (Staples et al, 2009). Mobile phases utilized were A: 0.1% formic acid, B: 50% acetonitrile and 0.1% formic acid.…”

Extended N-Sulfated Domains Reside at the Nonreducing End of Heparan Sulfate Chains

“…Oligosaccharide Analysis Using Amide-HILIC LC/MS-After desalting, lyase III-generated oligosaccharides were analyzed by amide-HILIC LC/MS as described previously (26,27). Briefly, the samples (0.8 g of lyase III-digested material) were injected onto a HILIC HPLC chip that was on line with an Agilent 6520 QTOF 6520 mass spectrometer operating in the negative ion mode.…”

Proteomic profiling of non-obese type 2 diabetic skeletal muscle

“…The sample was dried through vacuum centrifugation and the concentrated peptide fractions were then resuspended in mass spectrometry-grade distilled water and 0.1% formic acid for identification by ion trap LC-MS analysis. Separation of peptides was performed with a nanoflow Agilent 1200 series system, equipped with a Zorbax 300SB C18 5 μm, 4 mm 40 nl pre-column and an Zorbax 300SB C18 5 μm, 43 mm x 75 μm analytical reversed phase column using the HPLC-Chip technology (32).…”