Imaging mass spectrometry using a delay-line detector.

Microscope mode mass spectrometric imaging is crucially dependent on the availability of a high- resolution, position-resolved time-of-flight detector. Here, a new detection method for microscope mode imaging mass spectrometry is presented. A delay-line detector has been used for the first time as a position-sensitive detector in imaging mass spectrometry. The method is implemented on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF), as well as a secondary ion mass spectrometry time-of-flight (SIMS-ToF) instrument. Trypsinogen and bovine serum albumin samples have been used with a metal mask to determine the spatial resolution of the new detector using the MALDI-ToF instrument. The new detector set-up was successfully employed to generate mass resolved SIMS images from biological structures on the surface of thin tissue sections. The biological samples studied were taken from tumor grown from xenografted breast cancer cell lines and chicken embryonal sections.

Human gliosarcoma-associated ganglioside composition is complex and distinctive as evidenced by high-performance mass spectrometric determination and structural characterization.

Gangliosides (GGs), involved in malignant alteration and tumor progression/invasiveness, are considered as tumor biomarkers or therapeutic targets. Here, we describe the first systematic GG composition characterization in human gliosarcoma versus normal brain tissue using our recently developed mass spectrometry (MS) methods, based on nano-electrospray (nano-ESI), Fourier-transform ion cyclotron resonance (FT-ICR), and chip nano-ESI quadrupole time-of-flight (QTOF), complemented by thin-layer chromatographic (TLC) analysis and quantification. Combined MS enabled detection and structural assignment of 73 distinct GG species: many more than reported so far for investigated gliomas. Apart from the 7.4-times lower total GG content, gliosarcoma contained all major brain-associated species, however, in very altered proportions, exhibiting a highly distinctive pattern: GD3 (48.9%)>GD1a/nLD1>GD2/GT3>GM3>GT1b>GM2>GM1a/GM1b/nLM1>LM1>GD1b>GQ1b. MS also revealed abundant O-Ac-GD3; its sequencing provided structural evidence to postulate a novel O-Ac-GD3 isomer O-acetylated at the inner Neu5Ac-residue, previously not structurally confirmed. The high sensitivity and mass accuracy permitted the assignment of unusual minor species: GM4, Hex-HexNAc-nLM1, Gal-GD1, Fuc-GT1, GalNAc-GT1, O-Ac-GM3, di- O-Ac-GD3O-Ac-GD3, and O-Ac-GT3, not previously reported as glioma-associated. The gliosarcoma-expressed GA2 might represent a marker distinguishing astrocytic from oligodendroglial tumors. This is, to our knowledge, so far the most complete GG composition characterization of certain glioma, which demonstrates that our MS-based approach could provide essential structural information relevant to glycosphingolipid role(s) in brain tumor biology, differential diagnosis/prognosis and novel treatment concepts.

Screening and sequencing of complex sialylated and sulfated glycosphingolipid mixtures by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.

A protocol for negative ion nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (-)nanoESI-FTICR MS, investigation of complex biological mixtures consisting of sialylated or sulfated glycosphingolipids (GSL) expressing high heterogeneity in the ceramide portion is described. Different instrumental and solvent conditions were explored and optimized to promote efficient ionization, reduce the in-source fragmentation and consequently enhance the detection of intact molecular species from complex mixtures. Using the novel optimized (-)nanoESI-FTICR MS protocol, a reliable and detailed compositional fingerprint of the polysialylated ganglioside mixture isolated from human brain was obtained. Sustained off-resonance irradiation collision-induced dissociation mass spectrometry (SORI-CID MS2) was introduced for the first time for structural elucidation of polysialylated gangliosides. Under well-defined conditions, an informative fragmentation pattern of the trisialylated ganglioside GT1 was obtained. The compositional mapping of a complex mixture of sulfated glucuronic acid containing neolacto-series GSLs extracted from bovine Cauda equina provided hard evidence upon previously described components and new structures not identified before by any other analytical method. Negative ion nanoESI-FTICR MS at 9.4 T is shown here to represent a valuable method in glycolipidomics, allowing a high resolution and mass accuracy detection of major and minor GSL glycoforms and identification of known and novel biologically relevant structures.

Coupling of fully automated chip electrospray to Fourier transform ion cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing.

The NanoMate robot has been coupled to a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer at 9.4 T and implemented for the first time for complex carbohydrate analysis. It was optimized in the negative ion mode to achieve automated sample delivery on the chip along with increased sensitivity, ultra-high resolution and accurate mass determination. A novel bracket has been designed to allow a reliable mounting of the NanoMate to the Apollo electrospray ionization (ESI) source of an APEX II instrument. The notably higher efficiency of ionization for compositional mapping of complex mixtures and feasibility for fragmentation analysis of components by sustained off-resonance irradiation collision-induced tandem mass spectrometry (SORI-CID MS2) has been demonstrated on a glycoconjugate mixture containing O-glycosylated sialylated peptides from urine of a patient suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease), previously analyzed by capillary-based nanoESI-FTICRMS, and of a healthy control person. Due to its potential to generate highly charged ionic species, reduce the in-source fragmentation, increase sensitivity, reproducibility and ionization efficiency, along with the ability to generate a sustained and constant electrospray, this method can be considered as a new platform for advanced glycomics.

A thin chip microsprayer system coupled to Fourier transform ion cyclotron resonance mass spectrometry for glycopeptide screening.

A thin polymer microchip was coupled with a Fourier transform ion cyclotron resonance (FTICR) 9.4 T mass spectrometer and the method was optimized in negative ion mode for glycopeptide screening. The interface between the polymer microchip and FTICR mass spectrometer consists of an in-laboratory conceived and designed mounting system that exhibits robust and controllable alignment of the chip toward the inlet of the mass spectrometer. The particular attribute of the polymer chip coupled to the FTICR mass spectrometer, to achieve an increase in ionization efficiency and sensitivity under the premise of high mass accuracy of detection, is highlighted by the large number of major and minor glycopeptide structures detected and identified in highly heterogeneous mixtures obtained from urine matrices. Glycoforms expressing various saccharide chain lengths ranging from tri- to dodecasaccharide, bearing up to three sialic acid moieties, could be detected and assigned based on the accuracy of the mass measurement (average mass deviation below 6 ppm) of their molecular ions. -Thin chipESI-FTICRMS is a potent novel system for glycomic screening of complex mixtures, as demonstrated for identification of singly sialylated O-glycosylated amino acids and peptides from urine matrices, and could be considered for general applicability in the glycoanalytical field.

Sialylation analysis of O-glycosylated sialylated peptides from urine of patients suffering from Schindler's disease by Fourier transform ion cyclotron resonance mass spectrometry and sustained off-resonance irradiation collision-induced dissociation.

A strategy based on Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for screening of complex glycoconjugate mixtures containing O-linked glycopeptides and O-glycosylated amino acids with alpha-N-acetylgalactosaminyl residues is presented. To detect and identify O-glycoforms present in urine of patients suffering from hereditary N-acetylhexosaminidase deficiency (known as Schindler's disease), present at 100 times higher concentrations than in urine of healthy controls, new accurate methods for mapping and sequencing were required. In the mass spectrometric analysis particular attention has to be paid to original sialylation patterns, because of the potential lability of the sialic acid moiety during the desorption/ionization process. Negative ion nanoelectrospray ionization (nanoESI) FTICR-MS at 9.4 T is shown here to represent a method of choice for identification of single components in such complex glycomixtures due to high resolution and mass accuracy. By optimization of sustained off-resonance irradiation collision-induced dissociation tandem mass spectrometry (SORI-CID-MS(2)) in the negative ion mode, the type and sequence of the sialylated glycopeptide components were determined from their fragmentation patterns. Additionally, implementation of SORI-CID-MS(3) provides detailed information for sialylation analysis. The potential diagnostic value of this approach is discussed.