scholarly journals Benchmarking accuracy and precision of intensity‐based absolute quantification of protein abundances in Saccharomyces cerevisiae

PROTEOMICS ◽  
2021 ◽  
pp. 2000093
Author(s):  
Benjamín J. Sánchez ◽  
Petri‐Jaan Lahtvee ◽  
Kate Campbell ◽  
Sergo Kasvandik ◽  
Rosemary Yu ◽  
...  
Author(s):  
Benjamín J. Sánchez ◽  
Petri-Jaan Lahtvee ◽  
Kate Campbell ◽  
Sergo Kasvandik ◽  
Rosemary Yu ◽  
...  

AbstractProtein quantification via label-free mass spectrometry (MS) has become an increasingly popular method for determining genome-wide absolute protein abundances. A known caveat of this approach is the poor technical reproducibility, i.e. how consistent the estimations are when the same sample is measured repeatedly. Here, we measured proteomics data for Saccharomyces cerevisiae with both biological and inter-batch technical triplicates, to analyze both accuracy and precision of protein quantification via MS. Moreover, we analyzed how these metrics vary when applying different methods for converting MS intensities to absolute protein abundances. We found that a simple normalization and rescaling approach performs as accurately yet more precisely than methods that rely on external standards. Additionally, we show that inter-batch reproducibility is worse than biological reproducibility for all evaluated methods. These results subsequently serve as a benchmark for assessing MS data quality for protein quantification, whilst also underscoring current limitations in this approach.


PROTEOMICS ◽  
2021 ◽  
Vol 21 (15) ◽  
pp. 2170095
Author(s):  
Benjamín J. Sánchez ◽  
Petri‐Jaan Lahtvee ◽  
Kate Campbell ◽  
Sergo Kasvandik ◽  
Rosemary Yu ◽  
...  

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kanhaiya Kumar ◽  
Per Bruheim

Abstract Objective The objective of this study was to investigate the variation of NAD and CoA metabolite pools in Saccharomyces cerevisiae cultivated under various cultivation conditions. This study complements a previous report on glycolytic, pentose phosphate pathway, tricarboxylic acid cycle, amino acids, and deoxy-/nucleoside phosphate pools determined under the same cultivation conditions. Results S. cerevisiae pellets from batch (four carbohydrate sources) and chemostat (carbon-, nitrogen-, phosphate—limited and a range of dilution rates) bioreactor cultivations were extracted and analyzed with two recently established absolute quantitative liquid chromatography mass spectrometry (LC–MS/MS) methods for NAD and CoA metabolites. Both methods apply 13C internal standard dilution strategy for the enhanced analytical accuracy and precision. Individual metabolite pools were relatively constant for the different growth rates within the same mode of cultivation, but large differences were observed among some of the modes, i.e. NAD metabolites were 10 to 100-fold lower in nitrogen limited chemostats compared to the other modes, and phosphate limited chemostats were characterized with much lower CoA metabolite pools. The results complement the previous results and together provide a comprehensive insight into primary metabolite pools variations at a large range in growth and carbon source consumption rates.


2021 ◽  
Author(s):  
Davide Tamburro ◽  
Sinisa Bratulic ◽  
Souad Abou Shameh ◽  
Nikul K Soni ◽  
Andrea Bacconi ◽  
...  

AbstractGlycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.SummaryAnalytical performance of a kit for standardized GAG measurements, based on an established UHPLC-MS/MS method


2001 ◽  
Vol 36 (2) ◽  
pp. 196-201 ◽  
Author(s):  
F. Seibold ◽  
O. Stich ◽  
R. Hufnagl ◽  
S. Kamil ◽  
M. Scheurlen

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.


2007 ◽  
Vol 45 (08) ◽  
Author(s):  
S Schmechel ◽  
V Schachinger ◽  
F Seibold ◽  
C Tillack ◽  
T Ochsenkühn ◽  
...  

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