The progress of ion mobility spectrometry (IMS), together with its association to mass spectrometry (MS), opened new directions for the identification of various metabolites in complex biological matrices. However, glycolipidomics of the human brain by IMS MS represents an area untouched up to now, because of the difficulties encountered in brain sampling, analyte extraction, and IMS MS method optimization. In this study, IMS MS was introduced in human brain ganglioside (GG) research. The efficiency of the method in clinical glycolipidomics was demonstrated on a highly complex mixture extracted from a normal fetal frontal lobe (FL37). Using this approach, a remarkably rich molecular ion pattern was discovered, which proved the presence of a large number of glycoforms and an unpredicted diversity of the ceramide chains. Moreover, the results showed for the first time the occurrence of GGs in the human brain with a much higher degree of sialylation than previously reported. Using IMS MS, the entire series starting from mono- up to octasialylated GGs was detected in FL37. These findings substantiate early clinical reports on the direct correlation between GG sialylation degree and brain developmental stage. Using IMS CID MS/MS, applied here for the first time to gangliosides, a novel, tetrasialylated O-GalNAc modified species with a potential biomarker role in brain development was structurally characterized. Under variable collision energy, a high number of sequence ions was generated for the investigated GalNAc-GQ1(d18:1/18:0) species. Several fragment ions documented the presence of the tetrasialo element attached to the inner Gal, indicating that GalNAc-GQ1(d18:1/18:0) belongs to the d series.

Electron transfer dissociation (ETD) has been developed recently as an efficient ion fragmentation technique in mass spectrometry (MS), being presently considered a step forward in proteomics with real perspectives for improvement, upgrade and application. Available also on affordable ion trap mass spectrometers, ETD induces specific N-Cα bond cleavages of the peptide backbone with the preservation of the post-translational modifications and generation of product ions that are diagnostic for the modification site(s). In addition, in the last few years ETD contributed significantly to the development of top-down approaches which enable tandem MS of intact protein ions. The present review, covering the last 5 years highlights concisely the major achievements and the current applications of ETD fragmentation technique in proteomics. An ample part of the review is dedicated to ETD contribution in the elucidation of the most common posttranslational modifications, such as phosphorylation and glycosylation. Further, a brief section is devoted to top-down by ETD method applied to intact proteins. As the last few years have witnessed a major expansion of the microfluidics systems, a few considerations on ETD in combination with chip-based nanoelectrospray (nanoESI) as a platform for high throughput top-down proteomics are also presented.

We have developed here a superior approach based on high-resolution (HR) mass spectrometry (MS) for monitoring the changes occurring with development and aging in the composition and structure of cerebellar gangliosidome. The experiments were focused on the comparative screening and structural analysis of gangliosides expressed in fetal and aged cerebellum by Orbitrap MS with nanoelectrospray ionization (nanoESI) in the negative ion mode. The employed ultrahigh-resolution MS platform allowed the discrimination, without the need of previous separation, of 159 ions corresponding to 120 distinct species in the native ganglioside mixtures from fetal and aged cerebellar biopsies, many more than detected before, when MS platforms of lower resolution were employed. A number of gangliosides, in particular polysialylated belonging to GT, GQ, GP, and GS classes, modified by O-fucosylation, O-acetylation, or CH 3 COO − were discovered here, for the first time in human cerebellum. These components, found differently expressed in fetal and aged tissues, indicated that the ganglioside profile in cerebellum is development stage-and agespecific. Following the fragmentation analysis by high-energy collision-induced dissociation (HCD) tandem MS (MS/MS), we have also observed that the intimate structure of certain compounds has not changed during the development and aging of the brain, an aspect which could open new directions in the investigation of ganglioside biomarkers in cerebellar tissue.

General work-flow for ganglioside analysis by IM-MS.