Covaris

Automated Sample Preparation Combining Adaptive Focused
Acoustics® (AFA®) and Single Pot Solid Phase Sample Preparation (SP3)
Scientific Relevance
Sample preparation can significantly impact the quality of data in both academic and clinical research. Proteomics analytical methods
require robust, reproducible, and streamlined protocols [1,2]. This document presents an automated, high-throughput workflow yielding
highly reproducible results [3].
Challenges• Numerous transfer steps are involved		 in current proteomics sample prep• High-throughput and low volume• Some samples can be particularly		 processing is challenging		 difficult to process with		 workflows, increasing the risk for		 reproducibility		 sample loss, contamination, and errors
Workflow

Input
FFPE Scrolls, Fresh Frozen
Tissue, Cells

AFA Focused-ultrasonication & 95 °C Heating*
Lysis, Protein Extraction, DNA Shearing,
Emulsiﬁcation, and De-crosslinking*Reduction &Alkyation

Lysis and homogenization
steps are performed usinga Covaris instrument.

autoSP3
Reduction and Alkylation,
Protein Clean-up, and Digestion

Reduction, clean-up (orAdd SP3
Beads

purification), and digestion
are conducted using anProtein
Binding

3000
2500
2000

Agilent Bravo.

Washingon Magnet

LC-MS
Protein Identiﬁcation andQuantiﬁcation

The workflow efficiency
enables buffer flexibility,
3 x 80% EtOH
1 x 100% ACN

1500

Protein Digestion& Recovery

1000
500
0

2 x 80% EtOH
1 x 100% IsoP
1 x 100% ACN

including detergent-based
buffers (containingSDS up to 5%).

Optional:
Additional clean-up*Heating at 95 °C requires a block heater or a PCR instrument.

Advantages of the Covaris Workflow• Seamless integration of Adaptive Focused Acoustics with		 single pot processing: the sample remains in the same plate		 or strip throughout the whole process• Direct treatment of various sample types including FFPE orAdapted from and with courtesy of Jeroen Krijgsveld, DKFZ, Heidelberg

Suggested Products• Covaris Instruments: ML230, LE220Rsc, and R230
• Covaris Consumables: 8 AFA-TUBE TPX Strip,		
96 AFA-TUBE TPX Plate, and 384 AFA-TUBE 20 PP Plate• Agilent Bravo platform		 fresh tissue and cells• Full hands-off treatment and fast turn-around-time in		 combination with Agilent Bravo robot
References
1. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments – Hughes et al, Nat Protoc. 2019 Jan;14(1):68-85. DOI: 10.1038/s41596-018-0082-x
2. Ultrasensitive proteome analysis using paramagnetic bead technology, Hughes et al, Mol Syst Biol. 2014 Oct 30;10(10):757. DOI: 10.15252/msb.20145625
3. Automated sample preparation with SP3 for low‐input clinical proteomics, Mueller et al, Mol Syst Biol (2020)16:e9111 DOI: 10.15252/msb.20199111
Bravo® is a trademark of Agilent
Information subject to change without notice. For research only. Not for use in diagnostic procedures.

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