Ostro Protein Precipitation
This basic method provides the core methodology for translating a workflow into OneLab-executable script(s) as an attempt to fully or semi-automate a specific procedure. It demonstrates the benefits of automation and highlights OneLab capabilities and best practices to promote solution adoption, helping transition from manual to a more automated approach. It can be used alone or serves as a building block for a more complex workflow and is easily adaptable to users' requirements.
Overview
Automated Protein Precipitation Sample Prep for Higher Sensitivity and Precision
Sample preparation is a crucial step in an overall LC-MS bioanalysis workflow that is used to extract, purify, and concentrate samples from bio-matrices prior to analysis. Common sample preparation techniques include sample dilution, protein precipitation (PPT), liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Sample purification and cleanup steps are often required in order to achieve optimal sensitivity, selectivity, and accurate quantification during LC-MS analysis.
One of the simplest forms of sample cleanup is PPT. It involves taking a biological sample, diluting it with a protein disrupting solution (usually comprised of organic solvent), and extracting the analyte of interest from the matrix into the solution. This workflow may seem simple, however, it is common for errors to occur during this step due to the need for manual intervention (1). One such error that can occur is when the user is handling a pipette to transfer or dilute samples. The user can inadvertently pipette the wrong volume, dilute the wrong sample, or cause contamination by splashing during sample aspiration. Moreover, the need to reduce manual error is critical for the improvement of sample preparation workflows.
The Andrew+ Pipetting Robot can effectively carry out pipetting and extraction steps of a sample preparation workflow. Andrew+ runs on the cloud-native OneLab software to create methods for liquid handling and extraction of biological samples as well as initiate flow through a vacuum apparatus, all on one deck.
Automation Benefits
- Deliver accurate and precise pipetting
- Minimize user-to-user variability with transferrable methods
- Reduce the chance for human error and ensure data quality
- OneLab software provides a very intuitive graphical user interface for creating protocols, clear step-by-step guidance during execution, and full traceability
◼️ PROTOCOL 1 | Ostro PPT - Itaconic Acid | Andrew+
The work in protocol 1 demonstrates how the Andrew+ autonomously prepared a standard curve and QCs and carried out a simple pass-thru extraction method of itaconic acid from human plasma using the Waters Ostro PPT and phospholipid removal plate, for subsequent LC-MS analysis and quantification.
ORDERING INFORMATION
Andrew+ System Components: Dominos, Devices, Electronic Pipettes & Tips
- Deepwell Microplate Domino | p/n 186009597
- Microtube Domino | p/n 186009601
- 50mL Conical Centrifuge Tube Domino | p/n 186009614
- Storage Plate Domino | p/n 186009596
- 5mL Tip Rack Holder Domino | p/n 186009599
- 2x Tip Insertion System Domino | p/n 186009612
- Microplate Vacuum+ | p/n 176004579
- Andrew Alliance Bluetooth Electronic Pipette, 1-ch 300 μL | p/n 186009606
- Andrew Alliance Bluetooth Electronic Pipette, 1-ch 1000 μL | p/n 186009766
- Andrew Alliance Bluetooth Electronic Pipette, 1-ch 5000 μL | p/n 186009608
- Andrew Alliance Bluetooth Electronic Pipette, 8-ch 300 μL | p/n 186009607
- Sartorius, Optifit Tips, 5-350 μL | p/n 700013297
- Sartorius, Optifit Tips, 10-1000 µL | p/n 700013298
- Sartorius, Optifit Tips, 100-5000 µL | p/n 700013291
Consumables & Other Systems
- Ostro 96-square well sample preparation plate | p/n 186005518
- Waters 2 mL 96-square well collection plate, cut corner H1 | p/n 186002482
- Agilent 6-column reagent reservoir | p/n 201284-100
- Eppendorf 1.5 mL Safe-Lock tube | p/n 0030120086
- Falcon 50 mL conical centrifuge tube | p/n 352070
- Waters polypropylene cap mat for 96-well plate, square wells | p/n 186002484
- ACQUITY PREMIER HSS T3 column | p/n 186009468
- ACQUITY PREMIER system
- Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer
◼️ PROTOCOL 2 | Ostro PPT - Methylmalonic Acid | Andrew+
The work in protocol 2 shows the preparation and extraction of methylmalonic acid (MMA) in serum using the Waters Ostro PPT and phospholipid removal plate prior to LC-MS analysis.
ORDERING INFORMATION
Andrew+ System Components: Dominos, Devices, Electronic Pipettes & Tips
- 2x Deepwell Microplate Domino | p/n 186009597
- Microtube Domino | p/n 186009601
- Storage Plate Domino | p/n 186009596
- 2x Tip Insertion System Domino | p/n 186009612
- Microplate Vacuum+ | p/n 176004579
- Andrew Alliance Bluetooth Electronic Pipette, 1-ch 300 μL | p/n 186009606
- Andrew Alliance Bluetooth Electronic Pipette, 8-ch 300 μL | p/n 186009607
- Andrew Alliance Bluetooth Electronic Pipette, 8-ch 1200 µL | p/n 186009615
- Sartorius, Optifit Tips, 5-350 μL | p/n 700013297
- Sartorius, Optifit Tips, 50-1200 µL | p/n 700013300
Consumables & Other Systems
- Ostro protein precipitation & phospholipid removal 96-well plate | p/n 186005518
- Waters 2 mL 96-square well collection plate, cut corner H1 | p/n 186002482
- Agilent 6-column reagent reservoir | p/n 201284-100
- Axygen® 12-well reservoir with 12-channel trough | p/n RES-MW12-HP
- Fisherbrand Premium 1.5 mL microtube | p/n 11926955
- ACQUITY PREMIER HSS T3 column | p/n 186009468
- Microplate evaporator
- ACQUITY PREMIER system
- Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer
Figure 1: Simplified workflow for the PPT and phospholipid removal sample preparation and extraction method.
(1) Chapter 5 Automation tools and strategies for bioanalysis, in Progress in Pharmaceutical And Biomedical Analysis, David A. Wells, Editor 2003, Elsevier. p. 135-197.
Protocols
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This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 