Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-4 (of 4 Records) |
Query Trace: Andacht TM[original query] |
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The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses
Bennett KL , Wang X , Bystrom CE , Chambers MC , Andacht TM , Dangott LJ , Elortza F , Leszyk J , Molina H , Moritz RL , Phinney BS , Thompson JW , Bunger MK , Tabb DL . Mol Cell Proteomics 2015 14 (12) 3299-309 Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyse provided aliquots of a 6 bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of 8 LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyse the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intra-laboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9 month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the ABRF-PRG recommends that laboratories closely scrutinise the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable amongst many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114. |
An enhanced throughput method for quantification of sulfur mustard adducts to human serum albumin via isotope dilution tandem mass spectrometry
Andacht TM , Pantazides BG , Crow BS , Fidder A , Noort D , Thomas JD , Blake TA , Johnson RC . J Anal Toxicol 2013 38 (1) 8-15 Here, we report an enhanced throughput method for the diagnosis of human exposure to sulfur mustard. A hydroxyethylthioethyl (HETE) ester-adducted tripeptide, produced by pronase digestion of human serum albumin, was selected as the quantitative exposure biomarker. Cibacron Blue enrichment was developed from an established cartridge method into a 96-well plate format, increasing throughput and ruggedness. This new method decreased sample volume 2.5-fold. Addition of a precipitation and solid-phase extraction concentration step increased the sensitivity of the method. With the conversion to a 96-well plate and optimization of chromatography, the method resulted in a 3-fold decrease in analysis time. Inclusion of a confirmation ion has increased specificity. The method was found to be linear between 0.050 and 50 microM sulfur mustard exposure with a precision for both quality control samples of ≤6.5% relative standard deviation and an accuracy of >96%. The limit of detection (3So) was calculated to be approximately 0.0048 microM, an exposure value similar to that of the HETE-albumin adduct method first described by Noort and co-workers (Noort et al., 1999; Noort el al., 2004) which used protein precipitation to isolate albumin. A convenience set of 124 plasma samples from healthy unexposed individuals was analyzed using this method to assess background levels of exposure to sulfur mustard; no positive results were detected. |
The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses
Friedman DB , Andacht TM , Bunger MK , Chien AS , Hawke DH , Krijgsveld J , Lane WS , Lilley KS , Maccoss MJ , Moritz RL , Settlage RE , Sherman NE , Weintraub ST , Witkowska HE , Yates NA , Turck CW . Proteomics 2011 11 (8) 1371-81 Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices. |
Leptin modulated changes in adipose tissue protein expression in ob/ob mice
Zhang W , Ambati S , Della-Fera MA , Choi YH , Baile CA , Andacht TM . Obesity (Silver Spring) 2010 19 (2) 255-61 Comparative proteomic analyses were performed in adipose tissue of leptin-deficient ob/ob mice treated with leptin or control buffer in order to identify the protein expression changes as the potential targets of leptin. Mice were treated with either phosphate-buffered saline (control) or 10 microg/day leptin for 14 days via subcutaneous osmotic minipumps. Total protein from white adipose tissue was extracted and labeled with different fluorescent cyanine dyes for analysis by two-dimensional difference gel electrophoresis (DIGE). Spots that were differentially expressed and appeared to have sufficient material for mass spectrometry analysis were picked and digested with trypsin and subjected to MALDI-TOF MS for protein identification. Twelve functional protein groups were found differentially expressed in adipose tissue of leptin-treated vs. control ob/ob mice, including molecular chaperones and redox proteins such as calreticulin (CALR), protein disulfide isomerase-associated 3 (PDIA3), prohibitin (PHB), and peroxiredoxin-6 (PRDX6); cytoskeleton proteins such as beta actin, desmin, and alpha-tubulin; and some other proteins. The mRNA levels of CALR, PDIA3, and PHB were measured by real-time reverse transcription-PCR and found to be upregulated (P < 0.05), consistent with the fold change in protein expression level. Our findings suggest that leptin's effects on lipid metabolism and apoptosis may be mediated in part by alterations in expression of molecular chaperones and redox proteins for regulating endoplasmic reticulum stress and cytoskeleton proteins for regulating mitochondrial morphology. |
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