On-Line Nano-HPLC/ESI QTOF MS and Tandem MS for Separation, Detection, and Structural Elucidation of Human Erythrocytes Neutral Glycosphingolipid Mixture

Abstract

A superior approach involving nano-high-performance liquid chromatography (nano-HPLC) in on-line conjunction to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QTOF MS) and tandem MS for screening and structural characterization of complex mixtures of neutral glycosphingolipids (GSLs) is here described. Neutral GSLs purified from human erythrocytes were efficiently separated according to the differences in carbohydrate chain length by an optimized nano-HPLC protocol and flow-through detected by ESI QTOF MS at the low femtomole level. Additionally, GSL species were accurately distinguished from the accompanying lipids in the mixture, thus permitting the determination of detailed structural characteristics by data-dependent analysis for identification of GSL constitution within single experiments. An alternative nano-HPLC/ESI QTOF MS approach was designed for dissection of unsaturation/saturation degree of the ceramide moieties defining the hydrophobic portion of GSLs and subsequent localization by nano-HPLC/ESI QTOF MS/MS of the -CH=CH- within the ceramide regions. The method is fast, highly sensitive, and high-throughput amenable and is highlighted as a new and valuable analytical dimension in glycolipidomics.

Mass spectrometry of fluorocarbon‐labeled glycosphingolipids

“…2, Panel C), further sequential isolation and activation through the MS 4 pathway to www.interscience.wiley.com/journal/jms obtain fragments of the non-reducing terminal Galα4Gal-1-ene disaccharide at m/z 445 (1556 → 667 → 445 →) yielded a spectrum (Panel D) closely resembling that obtained previously from permethylated Gb 3 Cer, [12] and easily distinguishable from those obtained from terminal disaccharides with isomeric linkage structures, for example, the Galα3Gal-1-ene from iGb 3 Cer. Figure 1, Panel B, shows an HPTLC analysis of the reaction of SCDase with purified standard G M1 (generally regarded as G M1a , 5) and subsequent derivatization of the product with a typical F-tagging reagent, F9-Cbz-OSu, without intermediate isolation of the lyso-G M1 from the enzyme reaction.…”

“…Previous studies [12] established that permethylated Gb 3 Cer (Galα4Galβ4Glcβ1Cer) can be discriminated from iGb 3 Cer (Galα3Galβ4Glcβ1Cer) by ion trap CID-MS 4 MS via the pathway MNa + → 667 → 445 →. Starting from the Na + molecular adduct of permethylated F-tagged Gb 3 Cer, m/z 1556, products of the terminal disaccharide 1-ene fragment at m/z 445 (m/z 1556 → 667 → 445 →) were observed in a pattern almost indistinguishable from that of permethylated Gb 3 Cer, enabling automated identification by submission of the spectrum to a prototype fragment library (Fraglib) search tool, which compares it with data sets acquired by prior analysis of standard compounds under similar condition. [54] We have further developed a quantitative approach to iGb 3 Cer/Gb 3 Cer detection, [12] and in preliminary experiments found prior F-tagging protocols to be compatible with this method (Li et al, unpublished data).…”

“…1, Panel C, the spots of F-tagged G M1 were detected in a concentration dependent manner. This preliminary experiment showed that F-tagging could potentially provide an alternative format for generation of GSL microarrays, which would be even more useful if it were compatible with other useful analytical protocols, such as the permethylation and ion trap MS n analysis illustrated above for Gb 3 Cer.…”

“…[46] RBC Gb 3 Cer is known to be almost homogeneous with respect to sphingoid (d18 : 1), but heterogeneous with respect to fatty-N-acylation, as exemplified by the HPTLC analysis of . In cases where the precise fatty acid composition is not an important issue, it is apparent that such ceramide heterogeneity could be a factor decreasing the overall sensitivity of detection by partitioning the molecular signal into multiple ions (this is not always the case [12] ).…”

Sphingolipidomics: a valuable tool for understanding the roles of sphingolipids in biology and disease

“…Lipid MS uses various ionization methods [e.g., electrospray ionization, (ESI) and matrix-assisted laserdesorption ionization (MALDI)] combined with triple quadrupole or tandem quadrupole-linear ion trap mass analyzers for MS/MS and MS n (10), respectively, or for higher mass accuracy, time-of-flight (11) or Fourier transform instruments (12,13). The combination of MS with liquid chromatography (10,14), thin-layer chromatography (12,15), and automated microfluidic "chip"-based devices (16) has been successful in meeting difficult analytical challenges posed by the presence of contaminants that suppress ionization, isomeric species, etc.…”