Coupling of fully automated chip electrospray to Fourier transform ion cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing.
ABSTRACT 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.
SourceAvailable from: Adrian Cristian Robu[Show abstract] [Hide abstract]
ABSTRACT: In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as the control. 49 glycoforms were discovered in the investigated urine fraction from Schindler disease versus only 14 in control urine. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to serine, threonine, or threonine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). A number of 29 species discovered here, most of which with long chain glycans, were not previously reported as associated to this condition. All glycopeptides were detected in only 1min analysis time, with a sample consumption situated in the femtomole range.Carbohydrate Research 09/2014; 398. DOI:10.1016/j.carres.2014.08.014 · 1.97 Impact Factor
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ABSTRACT: Membrane proteomic analysis has been proven to be a promising tool for identifying new and specific biomarkers that can be used for prognosis and monitoring of various cancers. Membrane proteins are of great interest particularly those with functional domains exposed to the extracellular environment. Integral membrane proteins represent about one-third of the proteins encoded by the human genome and assume a variety of key biological functions, such as cell-to-cell communication, receptor-mediated signal transduction, selective transport, and pharmacological actions. More than two-thirds of membrane proteins are drug targets, highlighting their immensely important pharmaceutical significance. Most plasma membrane proteins and proteins from other cellular membranes have several PTMs; for example, glycosylation, phosphorylation, and nitrosylation, and moreover, PTMs of proteins are known to play a key role in tumor biology. These modifications often cause change in stoichiometry and microheterogeneity in a protein molecule, which is apparent during electrophoretic separation. Furthermore, the analysis of glyco- and phosphoproteome of cell membrane presents a number of challenges mainly due to their low abundance, their large dynamic range, and the inherent hydrophobicity of membrane proteins. Under pathological conditions, PTMs, such as phosphorylation and glycosylation are frequently altered and have been recognized as a potential source for disease biomarkers. Thus, their accurate differential expression analysis, along with differential PTM analysis is of paramount importance. Here we summarize the current status of membrane-based biomarkers in various cancers, and future perspective of membrane biomarker research.Proteomics 10/2012; 12(19-20):3085-104. DOI:10.1002/pmic.201100519 · 3.97 Impact Factor
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ABSTRACT: The presented study describes an adaptation of enzymatic assays to miniaturized and automated nanoelectrospray ionization (nanoESI)-mass spectrometry (MS). The system consists of a liquid handling robot including an ESI chip source and offers several advantages for the investigation of enzymatic reactions. Therewith different parameters can be tested rapidly and automatically. Thus the technique provides a basis for the efficient development of enzymatic assays using MS detection. In the present project the miniaturized setup was applied to become appropriate for the investigation of various enzymatic assays since real-time measurements using automated nanospray robots are not reported in detail so far. The reaction of hen egg white lysozyme (HEWL) and the saccharide hexa-N-acetylchitohexaose (GlcNAc)6 was used as an exemplary model system. The generation of a stable nanoelectrospray using aqueous solution with enzymes/proteins was typically the main difficulty. Thus various parameters were systematically tested and adjusted to overcome these instabilities. This resulted in moderate, but improved, spray stability. Moreover, the new adjustments were applied to further enzymatic assays (chitinase, chymotrypsin and acetylcholinesterase). In doing so, they were tested whether they are applicable in universal manner. These assays reveal additional findings like a partial reduction of enzymatic activity in the nanoESI setup. The observed complicacies in spraying pure aqueous solutions and the partial enzyme inactivation will be discussed in detail as well as possible approaches to overcome them.Analytical methods 04/2011; 3(4):822-830. DOI:10.1039/C0AY00727G · 1.94 Impact Factor