Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Gram Negative ◽  
Protein Quantitation ◽  
System P ◽  
Automated Protocol ◽  
Chloroform Methanol ◽  
Extract Protein

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

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v1

2020 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Gram Negative ◽  
Protein Quantitation ◽  
System P ◽  
Automated Protocol ◽  
Chloroform Methanol ◽  
Extract Protein

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

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Protein Quantitation ◽  
System P ◽  
Chloroform Methanol

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

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Protein Quantitation ◽  
System P ◽  
Chloroform Methanol

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

Automated Protein Quantification with the Biomek-FX Liquid Handler System v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Tad Ogorzalek ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Protein Quantification ◽  
Tryptic Digestion ◽  
System P ◽  
Plate Reader ◽  
Separate Control ◽  
Control Plate ◽  
Chloroform Methanol

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Automated Protein Quantification with the Biomek-FX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Tad Ogorzalek ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Protein Quantification ◽  
Tryptic Digestion ◽  
System P ◽  
Plate Reader ◽  
Separate Control ◽  
Control Plate ◽  
Chloroform Methanol

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Modular automated bottom-up proteomic sample preparation for high-throughput applications v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Tad Ogorzalek ◽  
Paul D. Adams ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Tryptic Digestion ◽  
Gram Negative Bacteria ◽  
Bottom Up ◽  
Preparation Methods ◽  
Gram Negative ◽  
E Coli ◽  
Protocol Development ◽  
Set Up

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

scholarly journals Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4698
Author(s):  
Sorel Tchewonpi Sagu ◽  
Gerd Huschek ◽  
Thomas Homann ◽  
Harshadrai M. Rawel
Keyword(s):  
Sample Preparation ◽  
Extraction Method ◽  
Trichloroacetic Acid ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Internal Standards ◽  
Allergenic Protein ◽  
Microwave Assisted ◽  
Allergenic Proteins ◽  
Detection And Quantification

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Modular automated bottom-up proteomic sample preparation for high-throughput applications v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Tad Ogorzalek ◽  
Paul D. Adams ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Tryptic Digestion ◽  
Gram Negative Bacteria ◽  
Bottom Up ◽  
Preparation Methods ◽  
Gram Negative ◽  
E Coli ◽  
Protocol Development ◽  
Set Up

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

Development of a C12Im-Cl-assisted method for rapid sample preparation in proteomic application

2021 ◽  
Author(s):  
Chang Liu ◽  
Xiaoxia Si ◽  
Shumei Yan ◽  
Xinyuan Zhao ◽  
Xiaohong Qian ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Sample Processing ◽  
Processing Methods ◽  
Proteomic Analyses

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods, including protein extraction and digestion, before MS remain as bottlenecks in the...

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