The Effects of Possible Contamination on the Radiocarbon Dating of the Dead Sea Scrolls II: Empirical Methods to Remove Castor Oil and Suggestions for Redating

While kept at the Rockefeller Museum in East Jerusalem, many Dead Sea Scroll fragments were exposed to castor oil by the original team of editors in the course of cleaning the parchments. Castor oil must be regarded as a serious contaminant in relation to radiocarbon dating. If modern castor oil is present and is not removed prior to dating, the 14C dates will be skewed artificially towards modern values. Earlier, it was shown that the standard AAA pretreatment procedure used in the 2 previous studies dating Dead Sea Scroll samples is not capable of removing castor oil from parchment samples. In the present work, we show that it is unlikely that castor oil reacts with the amino acids of the parchment proteins, a finding which leaves open the possibility of devising a cleaning method that can effectively remove castor oil. We then present 3 different pretreatment protocols designed to effectively remove castor oil from parchment samples. These involve 3 different cleaning techniques: extraction with supercritical CO2, ultrasound cleaning, and Soxhlet extraction—each with their own advantages and disadvantages. Our data show that the protocol involving Soxhlet extraction is the best suited for the purpose of decontaminating the Dead Sea Scrolls, and we recommend that this protocol be used in further attempts to 14C date the Dead Sea Scrolls. If such an attempt is decided on by the proper authorities, we propose a list of Scroll texts, which we suggest be redated in order to validate the 14C dates done earlier.

Site-specific glycoprofiling of N-linked glycopeptides using MALDI-TOF MS : strong correlation between signal strength and glycoform quantities

Site-specific glycoprofiling of N-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, but its quantitative accuracy lacks documentation. Thus, a systematic study of widely different glycopeptides was performed to determine the relationship between the relative abundances of the individual glycoforms and the MALDI-TOF MS signal strength. Glycopeptides derived from glycoproteins containing neutral glycans (ribonuclease B, IgG, and ovalbumin) were initially profiled and yielded excellent and reproducible quantitation (correlation coefficient r = 0.9958, n = 5) when evaluated against a normal phase HPLC 2-AB glycan profile. Similarly, precise quantitation was observed for various forms of N-glycans (free, permethylated, and fluorescence-labeled) using MS. In addition, three different sialoglycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids ( r = 0.9664, n = 5). For glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format proved capable of performing desalting without loss of quantitative information, but highlighted the column capacity as a critical parameter. In conclusion, MALDI-TOF MS signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, providing that certain technical issues are considered, i.e., nonbiased sample handling, matrix choice, and instrumental settings. This enables rapid and sensitive site-specific glycoprofiling of N-glycan populations to promote biomarker discovery and elucidation of glycan structure/function relationships.11 page(s

Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics

Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and nonglycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP2% FA ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP0.1% TFA and IP1% TFA ZIC-HILIC SPE) all containing 80% acetonitrile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC−ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP2% FA conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 nonglycosylated peptides observed in the IP2% FA ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE.12 page(s

Screening for Transglutaminase-Catalyzed Modifications by Peptide Mass Finger Printing Using Multipoint Recalibration on Recognized Peaks for High Mass Accuracy

Detection of posttranslational modifications is expected to be one of the major future experimental challenges for proteomics. We describe herein a mass spectrometric procedure to screen for protein modifications by peptide mass fingerprinting that is based on post-data acquisition improvement of the mass accuracy by exporting the peptide mass values into analytical software for multipoint recalibration on recognized peaks. Subsequently, the calibrated peak mass data set is used in searching for modified peptides, i.e., peptides possessing specific mass deviations. In order to identify the location of Lys- and Gln-residues available for transglutaminase-catalyzed isopeptide bond formation, mammalian small heat shock proteins (sHsps) were screened for labeling with the two hexapeptide probes GQDPVR and GNDPVK in presence of transglutaminase. Peptide modification due to cross-linking of the GQDPVR hexa-peptide probe was detected for C-terminal Lys residues. Novel transglutaminase-susceptible Gln sites were identified in two sHsps (Q31/Q27 in Hsp20 and HspB2, respectively), by cross-linking of the GNDPVK hexapeptide probe. Deamidation of specific Gln residues was also detected, as well an isopeptide derived from intramolecular Gln-Lys isopeptide bond formation. We conclude that peptide mass fingerprinting can be an efficient way of screening for various posttranslational modifications. Basically any instrumentation for MALDI mass spectrometry can be used, provided that post-data acquisition recalibration is applied

Alpha-Synuclein filaments bind the transcriptional regulator HMGB-1

Abnormal accumulation of alpha-synuclein filaments in Lewy bodies is a neuropathological hallmark of Parkinson's disease and sequestration of cellular protein into these protein aggregates may contribute to the degenerative process. We identified the transcriptional co-factor high mobility group protein 1 (HMGB-1) as a ligand for alpha-synuclein filaments by a filament spin-down technique, mass spectrometric peptide mapping and immunoblotting. HMGB-1 binds preferentially to aggregated alpha-synuclein and is present in alpha-synuclein filament-containing Lewy bodies isolated from brain tissue affected with dementia with Lewy bodies or Parkinson's disease. Our results demonstrate that alpha-synuclein filaments hold the potential for disturbing the cellular gene expression as they can sequester a component involved in cellular transcription regulation

Proteasomal inhibition by alpha-synuclein filaments and oligomers

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