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Automated Protein Extraction from Clinical Samples with theCovaris ML230 Focused-ultrasonicator
Authors: Manuela Staeber1, Daniela Ludwig2, Anja Freiwald1,2, Sameer Vasantgadkar3, Eugenio Daviso3, Nicolas Autret3, Markus Ralser2, and Michael Mülleder1
Affiliations: 1 - Core Facility High Throughput Mass Spectrometry, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin andHumboldt-Universität zu Berlin, Berlin 10117, Germany, 2 - Institute of Biochemistry, Charité – Universitätsmedizin Berlin, Corporate Member of FreieUniversität Berlin and Humboldt-Universität zu Berlin, Berlin 10117, Germany, and 3 - Covaris, Inc., Woburn, Massachusetts 01801
Keywords: Proteomics, FFPE, Fresh Tissue, Cells, SP3, and LC-MS

Abstract
Many research projects in translational or clinical laboratories require automated, hands-off solutions for protein sample preparation
which enable better reproducibility, increased efficiency, higher quality results, and faster turnaround time. This application notepresents protocols for simultaneous multi sample processing from diverse inputs in a convenient 8-strip format. Solutions aredescribed for protein extraction from formalin-fixed and paraffin-embedded (FFPE) tissue, fresh frozen tissue, and cultured cells
for mass spectrometry-based (MS) proteomics using short gradient runs. These protocols have been developed on the recently
launched Covaris ML230 Focused-ultrasonicator and optimized for a Single Pot Solid Phase Sample Preparation (SP3) workflow. The
same extraction workflows can be adapted applying other clean-up methodologies as well as different downstream analyses such as
western blotting or ELISA. The workflow presented here using the Covaris ML230 Ultrasonicator provides an ideal solution for midthroughput protein extraction with batch-sizes around 40 protein extractions per day.

Introduction

those protocols to our most recently released instrument, theSample preparation significantly impacts the data quality derived

Covaris ML230 Focused-ultrasonicator: this compact device isfrom the sample of interest including model systems as wellable to process single tubes as well as strips in a simultaneous

as primary clinical samples. This is especially relevant andmanner, offering the perfect solution for processing up to 40

important in the field of proteomics research, as proteins are an

samples per day.

extremely diverse community of macromolecules, present in an

Materials

extraordinarily wide range of concentrations, and they cannot be

Required Equipment

amplified. Many methods and protocols to isolate proteins areCovaris

available, of which Covaris Adaptive Focused Acoustics® (AFA®)• Covaris ML230 Focused-ultrasonicator (PN 500656)

has unique advantages towards:• AFA-TUBE TPX 8-strips (PN 520292) and caps (PN 500639)• Covering the diversity of samples (FFPE, LCM, fresh tissue,• PS ML230 Rack 8 AFA-TUBE TPX Strip (PN 500699)		 cells, bacteria, yeast…)• truXTRAC tissue lysis buffer (TLB) (PN 520284)• Reducing human error• Decreasing hands-on time

Other Suppliers• Increasing speed and throughput• Thermocycler• Significantly improving reproducibility and reliability• Magnetic Beads• Magnetic Rack

The efficiency of AFA for pre-analytical proteomics has
been demonstrated on numerous instruments, in various
conditions (volume, buffer, sample type) and throughputs (singleSamples

tubes, 8-strips, 96 and 384-well plates). Covaris Focused-

For autopsy informed, consent was given by the next of kin, andultrasonicators are proven to be an extremely robust andautopsies were performed on the legal basis of §1 SRegG BE of

flexible solution for mid- to high-throughput protein processing,

the autopsy act of Berlin. Animal material was obtained from anregardless of buffers and clean-up methods: it is fully efficientapproved study by the local animal ethics committee (LAGeSowith approaches like SP3 [1,2] or S-Trap [3]. Given the relevanceBerlin; T 0096/02) and carried out in accordance with EU Directiveof AFA in proteomics sample preparation workflows, we adapted

10/63/EU as well as in line with the ARRIVE guidelines.
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Methods
Protocols were adapted from previous collaborations on higher throughput instruments (LE220-plus and LE220R-plus) for fresh tissues andcells [2], and non-toxic FFPE [4] protein extraction. Those methods generally use SDS based buffers and SP3 as the clean-up and digestion
method. Other buffers [5] and other clean-up methods [3,6] can be used. You can contact applicationsupport@covaris.com or download
our Proteomics Quick Guide for up-to-date and optimized protocols.
Protein Extraction from FFPE Samples
Scrolls from human liver samples (1 to 2 mg FFPE tissue) were prepared and trimmed to remove excess paraffin. Sixteen samples wereprocessed in single wells of two 8 AFA-TUBE TPX Strips (Figure 1) filled with 75 µL (Strip 1) or 150 µL (Strip 2) of Covaris Tissue Lysis
Buffer (TLB), an SDS based buffer. A combination of heating (95 °C) and AFA (settings in Table 1) was used to reverse crosslinks andremove paraffin from the tissue samples. This unique and patented ultrasound-focused process allows paraffin to emulsify without any
organic solvent, resulting in a milky sample solution which can be processed further without intermediate filtration steps.

Volume (µL)

PIP (W)% DF

CPBRepeats

Pulse or Time
Continuous (s)Delay
Duration(s)Temp (°C)

Dither

75375

2550

201010183 mm Y dither @20 mm/s

150

350

25200

N/A

300

N/A

183 mm Y dither @20 mm/s

Table 1. AFA treatment settings for FFPE tissue deparaffinization and homogenization.

Different approaches exist for protein clean up. Amongst them, the SP3 protein aggregation [1] method allows for direct removal of allcontaminants and protein digestion and peptide release in the same vessel.
To remove paraffin and SDS and for subsequent protein purification, proteins are captured by magnetic beads followed by thorough alcohol
washes. Peptides are released in the supernatant through proteolytic on-bead digestion, thus avoiding additional transfer steps andkeeping the process ‘single-pot’ as described in other publications [2,4-6]. The standard protocol is depicted in Figure 1.
Add truXTRAC tissue lysis buﬀer
95 °C, 10 min, thermocycler
Covaris Deparaﬃnization (300s)
95 °C, 60 min, thermocycler
Covaris Homogenization (300 sec)

FFPE Scroll Samples

ML230 and 8 AFA-TUBE TPX Strip

Trypsin

LysC

MS-sample
Preparation

LC-MS/MSOn-bead Protein Capture

Figure 1. Workflow of protein extraction from FFPE in a 8-strip format. FFPE scrolls are dispensed in the 8-strip. TLB buffer is added, and samples areheated to 95 °C in a thermocycler. They undergo a first AFA treatment to emulsify the paraffin. A one-hour treatment at 95 °C allows to reverse crosslink
the tissue. A second AFA treatment is made to homogenize the tissue and release the proteins. Beads are added for protein capture and after several
washes the proteins are digested with trypsin. Adapted from Schweizer et al., Covaris application note M020141.
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Alkylation and reduction (TCEP/CAA) are combined in one step

Results

at 95 °C. On beads capture is performed under a 10:1 beadProtein extraction from FFPE samples

to protein ratio (1:1 hydrophilic/hydrophobic), with 50% ACN.

Peptides were quantified after digestion using Thermo

Different washes with ethanol are made, before a final acetonitrile

Fluorometric Kit resulting on average in 25 to 50 µg total quantity.

wash. Proteins are then reconstituted in ABC with 1:100 trypsin

Five micrograms of peptides were run on a 5 min gradient using

and digested over night at 37 °C.

scanning Swath (Agilent Infinity II - Sciex 6600 Q-TOF). For data
analysis, DIA-NN was used with an in-house project specificProtein Extraction from Fresh Tissue

spectral library, generated with gas-phase fractionation on Thermo

Twenty-four samples from the same rat liver (2 to 8 mg) wereQ-Exactive Plus [7-8].

collected in AFA-TUBE TPX 8-strips filled with 75 µL of lysis buffer(1% SDS in 0.1 M ammonium bicarbonate (ABC) + 1.25x protease

Results were analyzed at 1%FDR. Around 15,000 peptides

inhibitor cocktail). Samples are processed directly for protein(Figure 2) and 2,500 proteins groups were found for samplesextraction with one or two rounds of AFA treatment, depending

processed in 75 µL (Figure 3 right panel), and about 4% more foron the input. The same purification approach (SP3) described forboth for samples extracted in 150 µL (Figure 2 & 3, left panel),

FFPE samples (without deparaffinisation and de-crosslinking) issuggesting that 75 µL was not enough to fully cover the scroll andused for cleaning and digesting the proteins.

emulsify all the paraffin, resulting in some protein loss. Median
CV was found at 14.2% for 150 μL and 13.7% for 75 μL, for theLoad Tissue
intoAFA-TUBETissue
homogenization
75 µL
with AFA

overall workflows from protein extraction to raw data analysis, inProtein
Puriﬁcation Trypsin
Digestionand clean-up(e.g. SP3)

consistency with previous studies using AFA. Approximately 7%

Proteinready forprocessingof variance can be at attributed to sample digestion and LC_MS
analysis. Variance distribution across protein groups is very

AFA
Volume(µL)
75PIP
(W)
375

DF(%)
25

CPB Repeats

5020

conserved between the two sets of tested volumes.
Pulse or Time
Delay
Continuous (s) Duration(s)Pulsing
30*10sec

Temp(°C)

Dither123 mm Y
dither@ 20mm/s

10Precursors, 1%FDR
20000

15000

Table 2. AFA treatment settings for fresh tissue homogenization.

Protein Extraction from Cultured Cells

10000

Sixteen replicates of 400.000 HeLa cells were dispensed in 2 AFATUBE TPX 8-strips filled with 75 µL of lysis buffer (1% SDS in 0.15000

M ABC + 1.25x protease inhibitor cocktail). Samples are processed
directly for protein extraction with one round of AFA treatment.

0The same purification approach (SP3) described for fresh tissuesamples is used for cleaning and digesting the proteins.

A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 H2Figure 2. FFPE samples: precursor count at 1%FDR. Letters indicates the different
wells in strips 1 (75 µL) and 2 (150 µL).

Volume(µL)

PIP(W)DF(%)

CPB Repeats

75375

2550

50

Pulse or Time
Delay
Continuous (s) Duration(s)Pulsing
30*10sec

10Temp(°C)

Dither183 mm Y
dither
@ 20mm/s

Table 3. AFA treatment settings for cultured cells homogenization.

3Data analysisCovaris

fresApplication Note | www.covaris.com

human liver (FFPE)

humancount liver (FFPE)
150 µL

Precursor
filter onanalysis

des~25,000 75 µL
1% FDR
proteotypic peptides~17,500

fresh frozen tissue

countPeptides~25,000
1%Proteins
FDR~2,250
proteotypicpeptides
median CV
15 %~17,500

Literature
2.8k proteins in short runs
ann group)
Figure3. Variance distribution across protein groups (FFPE). More than 2,000~2,250

Figure 5. Variance distribution across protein groups (Fresh frozen).

proteins are quantified below 30% and almost
1,000 below10%DIA-FAIMS-LC-MS/MSvariance.
21 minwithV
15 %hort gradientsand specific library
MS/MS 2.8k proteins in shortBekker-Jensen
et al. 2020
runs

http://dx.doi.org/10.1074/mcp.TIR119.001906
21 min with DIA-FAIMS-LC-MS/MS
and specific library
Bekker-Jensen et al. 2020
http://dx.doi.org/10.1074/mcp.TIR119.001906
Zhu et al 2020
http://dx.doi.org/10.1002/1878-0261.12570

570

Figure 4. Pearson correlation coefficients across and between strips (FFPE)

Figure 6. Correlation analysis (Pearson) shows high reproducibility of the workflow.

Letters indicates the different wells in strips 1 (75 µL) and 2 (150 µL).

Hierarchical clustering separates strip 3 but fails to distingues Covaris strip 1 and 2.
Technical replicates for sample digestion and LC-MS cluster together (002,003,004).

Pearson correlations across all proteome measurements were highReproducibility was very good with median Calculated median CVwith values above 0.96, showing high workflow reproducibility

found at 15%. Like in the FFPE experiments, the consistency of(Figure 4). This correlation happens to be very high evenidentification between replicates was very high with all Pearson

between the two different volumes. Protein extraction from freshcorrelation values above 0.97 (Figure 6).

tissue Peptides were quantified after digestion using Thermo
Fluorometric Kit resulting on average in 30 to 100 µg total

Protein Extraction from Cultured Cells

quantity. Five micrograms of peptides were run on 5 min gradient

Peptides were quantified after digestion using Thermousing scanning Swath (Agilent Infinity II - Sciex 6600 Q-TOF). For

Fluorometric Kit resulting in up to 30 µg total quantity. Fivedata analysis, DIA-NN was used with an in-house project specificmicrograms of peptides were run on 5 min gradient using

spectral library, generated with gas-phase fractionation on Thermo

scanning Swath (Agilent Infinity II - Sciex 6600 Q-TOF). For data

Q-Exactive Plus.

analysis, DIA-NN was used with an in-house Hela spectral library,generated with gas-phase fractionation on Sciex 6600 Q-TOF.

Results were analyzed at 1%FDR. Around 17,500 peptides (notshown) and 2,250 proteins groups were identified (Figure 5), fullyin line with other similar studies [9].

4Covaris

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HeLa
Cells
HeLa Cells
average compared to conventional 2h gradient analysis. Longergradients have been used with successful results on mouse liver[10]. The extraction process is fully compatible with a single pot
approach, e.g. using SP3 to clean up and digest the proteins [2,10].

ptidesResults are highly comparable to those found on similar samples
processed on different Covaris instruments [2, 4, 11-12].
Here we show a fast, efficient, and reproducible mid-throughputprotein extraction workflow that is ideal for sample series of 10
to 40 samples. Consumables and workflows are highly reproducible.
Therefore, no or minimal batch effects are expected. This allows

Figure 7. Variance distribution across protein groups (HeLa cells).

onal analysis

preparation of hundreds of samples within a week and enables
mid-sized studies in pre-clinical and clinical research with, e.g.:• Samples from the clinic, such as fresh frozen tissue material• Samples from pathology or biobanks such as PFA, FFPE or		 DBS samples• Targeted assays for marker protein identification

References

1. Ultrasensitive proteome analysis using paramagnetic bead technology. CS		 Hughes et al., Mol Syst Biol. 2014;10:757. DOI: 10.15252/msb.20145625
2. Automated sample preparation with SP3 for low-input clinical proteomics.		 Mueller et al., Mol Syst Biol (2020)16:e9111. DOI: 10.15252/msb.20199111
3. Laser capture microdissection coupled mass spectrometry (LCM-MS) for		 spatially resolved analysis of formalin-fixed and stained human lung tissues.		 Herrera et al., Clin Proteom (2020) 17:24 DOI : 10.1186/s12014-020-09287-6
4. AFA-sonication Followed by Modified Protein Aggregation Capture (APAC) Enables		 Direct, Reproducible and Non-toxic Sample Preparation of FFPE Tissue for Mass		 Spectrometry based Proteomics. Schweitzer et al., Covaris Application note, https://		 www.covaris.com/wp/wp-content/uploads/2020/06/M020141.pdf
5. A high-efficiency cellular extraction system for biological proteomics. Dhabaria		 et al., J of Proteome Res. 2015 August 7; 14(8): 3403–340.		 DOI: 10.1021/acs.jproteome.5b00547
6. HYPERsol: High-Quality Data from Archival FFPE Tissue for Clinical Proteomics.		 Marchione et al., J. Proteome Res. 2020, 19, 2, 973–983.		 DOI: 10.1021/acs.jproteome.9b00686
7. Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. DIA-NN: neural 				 networks and interference correction enable deep proteome coverage in high		 throughput. Nat Methods. 2020;17: 41–44. DOI: 10.1038/s41592-019-0638-x
8. Messner CB, Demichev V, Bloomfield N, Yu JSL, White M, Kreidl M, et al. Ultra-fast		 proteomics with Scanning SWATH. Nat Biotechnol. 2021; 1–9.		 DOI: 10.1038/s41587-021-00860-4
9. Friedrich C, Schallenberg S, Kirchner M, Ziehm M, Niquet S, Haji M, et al.		 Comprehensive micro-scaled proteome and phosphoproteome characterization		 of archived retrospective cancer repositories. Nat Commun. 2021;12: .1–15.		 DOI: 10.1038/s41467-021-23855-w
10. Dietary-challenged mice with Alzheimer-like pathology show increased energy		 expenditure and reduced adipocyte hypertrophy and steatosis. Schreyer et al., Aging		 2021, Vol. 13, N°.8, P10891-10919. DOI: 10.18632/aging.202978
11. Paired liver biopsy and plasma proteomics study reveals circulating biomarkers for		 alcohol-related liver disease, Niu et al., biorxiv 2020.		 DOI: 10.1101/2020.10.16.337592
12. Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and		 SARS-CoV. Stukalov et al. , bioRxiv 2020. DOI: 10.1101/2020.06.17.156455

Figure 8. Pearson correlation coefficients (HeLa cells).

Results were analyzed at 1%FDR. Around 30,000 peptides (notshown) and 4,000 proteins groups were identified (Figure 7),
which was very satisfactory with regards to the gradient length,
and compatible with other studies [2]. Of interest, the calculated
median CV was a slightly higher (18.1%) when compared to theother samples run on the ML230 (Figure 6). An explanation camefrom the Pearson correlation analysis, with one sample (D2, Figure8) being clearly outside of the range of correlation found for all theother replicates. Without this sample, CV improves significantly toreach 13.9%.

Conclusions
The employed protein extraction workflow is easy to adapt tothe ML230 Covaris Focused-ultrasonicator and displays highly
consistent and reproducible results for the various sample inputs
tested (cultured cells, fresh tissue and FFPE samples). Peptideand protein identifications are very good for short gradients andInformation subject to change without notice. For research only. Not for use in diagnostic procedures.

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