Good Old-Fashioned Protein Concentration Determination by Amino Acid Analysis

May, Caroline; Serschnitzki, Bettina; Marcus, Katrin

doi:10.1007/978-1-0716-1024-4_2

Cited by 8 publications

(7 citation statements)

References 4 publications

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“…CSF datasets we generated used four digestion strategies, including standard in-solution digestion, rapid in-solution digestion, standard filter-aided sample preparation (FASP), and rapid FASP, assuming different sample preparations result in different peptide recovery after digestion, leading to variations at the peptide and protein levels. To ensure that the same amount of peptides was analyzed using MS for all the samples, amino acid analysis (AAA) [ 44 ] had been performed for peptide quantification. The peptide quantification results already confirmed that the different digestion methods vary in sample recovery.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The peptide concentration in the digested samples was determined by amino acid analysis (AAA) as described by Plum [ 43 ] and May et al [ 44 ]. According to AAA, 500 ng per sample was taken for nanoLC-MS analysis and analyzed as previously described [ 43 , 44 ]. Briefly, extracted peptides were first injected and pre-concentrated with an UltiMate™ 3000 RSLCnano system (Thermo Fisher Scientific Inc., Waltham, MA, USA) using a trap column (Acclaim PepMap 100, 300 μm × 5 mm, C18, 5 μm, 100 Å; flow rate 30 μL/min).…”

Section: Methodsmentioning

confidence: 99%

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Quality Control—A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome

et al. 2023

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Proteomic studies using mass spectrometry (MS)-based quantification are a main approach to the discovery of new biomarkers. However, a number of analytical conditions in front and during MS data acquisition can affect the accuracy of the obtained outcome. Therefore, comprehensive quality assessment of the acquired data plays a central role in quantitative proteomics, though, due to the immense complexity of MS data, it is often neglected. Here, we address practically the quality assessment of quantitative MS data, describing key steps for the evaluation, including the levels of raw data, identification and quantification. With this, four independent datasets from cerebrospinal fluid, an important biofluid for neurodegenerative disease biomarker studies, were assessed, demonstrating that sample processing-based differences are already reflected at all three levels but with varying impacts on the quality of the quantitative data. Specifically, we provide guidance to critically interpret the quality of MS data for quantitative proteomics. Moreover, we provide the free and open source quality control tool MaCProQC, enabling systematic, rapid and uncomplicated data comparison of raw data, identification and feature detection levels through defined quality metrics and a step-by-step quality control workflow.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Quality Control—A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome

et al. 2023

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“…For amino acid analysis, each sample was dried completely in a glass tube. Subsequent amino acid analysis was performed using the Acquity HPLC and AccQ-Tag Ultra system (Waters GmbH) as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

Case Report: Muscle Wasting during Severe Sustained Hypoxia in Two Professional Mountaineers

Pesta

Heieis

Hand

et al. 2022

Medicine &Amp; Science in Sports &Amp; Exercise

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Purpose: Chronic exposure to hypoxia can induce muscle wasting in unaccustomed individuals. Detailed assessment of the effects of hypoxia on muscle tissue adaptation in elite mountaineers has not been performed. This study aims to assess muscle volume after exposure to normobaric hypoxia. Methods: Two professional mountaineers (A and B) participated in a 35-d intervention of graded normobaric hypoxia with the aim of 14 d exposure to 8% oxygen corresponding to 7112-m altitude. Volume of the shank, thigh, and hip muscles was assessed by magnetic resonance imaging pre-and postintervention. Dietary intake and physical activity were monitored throughout the study from food images and accelerometry analysis, together with blood analysis and anthropometric measurements. Results: Hypoxia reduced total leg muscle volume by 3.3% ± 6.0% in A and by 9.4% ± 7.3% in B. A lost 288 g and B 642 g of muscle mass, whereas dietary intake only declined by ~23% in the last intervention week. Arterial oxygen saturation declined from 95% and 86% to 77% and 72% in A and B, respectively. In hypoxia, participants could not maintain their physical activity levels. Notably, muscle loss varied substantially across muscle groups amounting to 5.4% ± 3.0%, 8.3% ± 5.2%, and 4.1% ± 8.6% for hip, thigh, and shank muscles, respectively. Conclusions: Our results indicate that hypoxia and resultant reductions in physical activity and caloric intake lead to substantial loss of muscle mass that was accentuated in proximal muscle as opposed to distal muscles. Surprisingly, thigh muscle wasting during this intervention is comparable with that observed during strict 56-d bed rest.

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“…Samples were dried in a vacuum concentrator and resuspended in 40 µl 0.1% (v/v) TFA. Afterwards, the peptide concentration was determined by amino acid analysis (AAA) as described previously 31 .…”

Section: Mass Spectrometry For the Identification Of Atad1 Interactio...mentioning

confidence: 99%

Peroxisomal ATPase ATAD1 acts in quality control of the protein import machinery

Ott

Sehr

Lindemann

et al. 2022

Preprint

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ATAD1 is an AAA-ATPase which shows a dual localization at mitochondria and peroxisomes. While its peroxisomal function is not known, in mitochondria ATAD1 is part of a quality control mechanism extracting mislocalised tail-anchored and accumulated precursor proteins from the outer membrane. Here, we studied the peroxisomal interactome of ATAD1 and could show that human ATAD1 interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins which transiently inserts into peroxisomal membranes. Upon cargo-release, mono-ubiquitinated PEX5 is recycled into the cytosol by the AAA-peroxins PEX1 and PEX6. The accumulation of ubiquitinated PEX5 is known to trigger degradation of whole organelles called pexophagy. Here, we used ATAD1-, PEX1- and ATAD1/PEX1-CRISPR-Knockout cell lines to investigate the physiological role of an ATAD1-PEX5 interaction. We could show an influence of ATAD1 on the stability of accumulated PEX5 and hypothesize a role in a peroxisomal quality control mechanism enabling clearance of ubiquitinated receptor from the membrane.

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Good Old-Fashioned Protein Concentration Determination by Amino Acid Analysis

Cited by 8 publications

References 4 publications

Quality Control—A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome

Quality Control—A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome

Case Report: Muscle Wasting during Severe Sustained Hypoxia in Two Professional Mountaineers

Peroxisomal ATPase ATAD1 acts in quality control of the protein import machinery

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