11 research outputs found
Identification of Novel Ī±-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method
Parkinsonās disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated Ī±-synuclein (Ī±-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of Ī±-syn in brain tissue homogenates. N-terminally acetylated full-length Ī±-syn (Ac-Ī±-syn1ā140) and two N-terminally acetylated C-terminally truncated forms of Ī±-syn (Ac-Ī±-syn1ā139 and Ac-Ī±-syn1ā103) were found. The different forms of Ī±-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of Ī±-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA)
J Proteome Res
The neurodegenerative disorder Alzheimerās disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies
In vivo stable isotope labeling by amino acids in Drosophila melanogaster
The fruit fly Drosophila melanogaster is one of the most widely used and well-studied model organisms in biology and therefore a promising tool for quantitative proteomics. Here, we describe a method to label D. melanogaster with stable isotope labeled amino acids in vivo. Feeding flies with heavy lysine labeled yeast cells leads to virtually complete heavy labeling already in the first filial generation. The approach is simple, fast, and cost-effective, which makes SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics
Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid Ī²-peptides in human cerebrospinal fluid
The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid Ī² (AĪ²)-peptides, e.g., AĪ²1-42, is considered a critical step in the pathogenesis of Alzheimerās disease (AD). Although APP is a well-known membrane glycoprotein carrying both N- and O-glycans, nothing is known about the occurrence of released APP/AĪ² glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified AĪ² peptides and glycopeptides from CSF samples and then liquid chromatographyātandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/AĪ² peptides, we identified 37 APP/AĪ² glycopeptides with sialylated core 1 like O-glycans attached to Thr(ā39, ā21, ā20, and ā13), in a series of APP/AĪ²X-15 glycopeptides, where X was ā63, ā57, ā52, and ā45, in relation to Asp1 of the AĪ² sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the AĪ²1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)1-2Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5AcĪ±2,8Neu5Ac linkage. We could not detect any glycosylation of the AĪ²1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated AĪ² peptides in CSF in six AD patients compared to seven non-AD patients. APP/AĪ² sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD
Rapid and Effective Focusing in a Carrier Ampholyte Solution Isoelectric Focusing System: A Proteome Prefractionation Tool
Mass spectrometry in high-throughput proteomics: ready for the big time
Mass spectrometry has evolved and matured to a level where it is able to assess the complexity of the human proteome. We discuss some of the expected challenges ahead and promising strategies for success