protein quantitation
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2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System


2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system


2021 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system


2020 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System


Author(s):  
EmmaRae L. Murphy ◽  
Andrew P. Joy ◽  
Rodney J. Ouellette ◽  
David A. Barnett
Keyword(s):  

Author(s):  
Constance A. Sobsey ◽  
Robert Popp ◽  
Sahar Ibrahim ◽  
Bjoern C Froehlich ◽  
Adriana Aguilar-Mahecha ◽  
...  

2020 ◽  
Vol 157 ◽  
pp. 104954
Author(s):  
Tingting Hu ◽  
Kangle Zheng ◽  
Ping SU ◽  
Yi Yang ◽  
Liang Li ◽  
...  

Author(s):  
Jesse G. Meyer

ABSTRACTShotgun proteomics techniques infer the presence and quantity of proteins using peptide proxies, which are produced by cleavage of all isolated protein by a protease. Most protein quantitation strategies assume that multiple peptides derived from a protein will behave quantitatively similar across treatment groups, but this assumption may be false for biological or technical reasons. Here, I describe a strategy called peptide correlation analysis (PeCorA) that detects quantitative disagreements between peptides mapped to the same protein. Simple linear models are used to assess whether the slope of a peptide’s change across treatment groups differs from the slope of all other peptides assigned to the same protein. Reanalysis of proteomic data from primary mouse microglia with PeCorA revealed that about 15% of proteins contain one discordant peptide. Inspection of the discordant peptides shows utility of PeCorA for direct and indirect detection of regulated PTMs, and also for discovery of poorly quantified peptides that should be excluded. PeCorA can be applied to an arbitrary list of quantified peptides, and is freely available as a script written in R.


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