From Tissues to LC-MS-ready Protein Digests:
Automated Sample Preparation Enabled by Adaptive®
®
Focused Acoustics (AFA ) TechnologyCovaris

www.covaris.com

Authors: Patrick McCarthy1, Rachel Muriph2, Luca Beker2, Nicolas Autret1, Jim Laugharn1, Jason Evans2, and Ulrich Thomann1
Affiliations: 1 - Covaris, LLC., Woburn, Massachusetts 01801 and 2 – University of Massachusetts College of Science and Mathematics

Background and Introduction

Results

LC-MS based analysis of peptide hydrolysates obtained from digested proteins is important

The AFA-enhanced PAC workflow was optimized using commercial K562 protein extract. Afterfor pre-clinical evaluation of compound libraries, as well as for discovery and analysis ofproteins were reduced and alkylated, AFA-inferred mixing was used to enhance bead-binding.

biochemical pathways in translational medicine. While the last couple of decades witnessed

Non-selective (total protein) binding with AFA was similar to that observed with thermomixer,

tremendous growth in analytical technology such as high-resolution accurate massconferring a small advantage to binding efficiency (~2 - 8 % improvement; data not shown).

spectrometry, liquid chromatography, and informatics (enabling workflows from data analysis
to data management), lagging progress in sample extraction from biological matrices andOn-bead digestion experiments were performed with 1:10 or 1:50 trypsin ratios. On-beadpurification of analytes often results in inefficient and poor-quality data, regardless of the end-trypsin digestion was done by using either a thermomixer for 1 and 16 hours or AFA for 1

detection analytical tools. Here, we introduce a comprehensive and fully automatable sample

hour. AFA-enhanced mixing resulted in increased protein and peptide IDs as compared to bothpreparation workflow that incorporates Adaptive Focused Acoustics (AFA)-enabled efficient

1 and 16 hour heat block controls (Figure 3). The analysis of the digests was performed oncell lysis, protein extraction, protein/bead binding, and on-bead trypsin digestion. Thisa Fusion Lumos Tribrid Mass Spectrometer coupled to an Easy nLC 1200 HPLC system. The

workflow requires only three liquid transfer steps, resulting in a robust process anddata was analyzed using Proteome Discoverer.

guaranteeing high quality data.
Adherent cell detachment workflows were compared using either trypsinization or AFA torecover HCT 116 cells from polystyrene culture plates. The AFA method is faster, fully

Adaptive Focused Acoustics (AFA) Technology

automatable, and significantly more user friendly than the trypsin method (Table 1).
The full workflow, starting from adherent cell culture was done with HCT 116 cells.  Following
detachment by either AFA or trypsinization, cells were transferred into a Covaris 96 AFATUBE TPX 150 Plate, subjected to AFA-based lysis and DNA shearing, and prepared for AFAenhanced trypsin digestion for 1 hour (Figure 4). Cells displaced with AFA resulted in a similar
number of protein and peptide IDs as compared to standard trypsinization.
A.

oscillate, and collapse.

• Acoustic energy can be adapted and shear forces can be low to gently mix samples or		

keep magnetic beads in suspension, but also high enough to disrupt biomolecule		complexes or even fragment macro molecules.

MethodSuspension and adherent cells were grown in standard 96-well polystyrene culture plates(Corning) following ATCC culturing recommendations.
The following workflow is fully automatable: Suspension and adhesive cultures werewashed with 1X PBS. To detach adherent cells, 40 µL of PBS per well was added and plates
were subjected to AFA in the R230 Focused-ultrasonicator. AFA energy settings have been

B.

2450
2400
2350
2300
2250
2200
2150
2100
2050

K562 PEPTIDE IDENTIFICATIONSPEPTIDE ID'S

• Fluctuations cause dissolved gasses to form microscopic bubbles​that grow,		

2500
PROTEIN ID'S

• Ultra-high frequency electronics and transducers produce and focus acoustic waves.

PROTEINS IDENTIFIED: K562 PROTEIN EXTRACT

Heated Shaker(16 hours)

Heated Shaker(1 hour)

R230 - AFA(1 hour)

1:10

2310

2347

2442

1:50

2326

2240

2371

9000
8000
7000
6000
5000
4000
3000
2000
1000
02

10Total Peptides

MISSED CLEAVAGES
Heated Shaker (16 hours)

Heated Shaker (1 hour)

R230 - AFA (1 hour)

Figure 3. LC-MS Analysis of Tryptic Digestion of K562 Cell Protein Extract. K562 protein extract was bound to magnetic beads
using AFA-enhanced mixing and then digested with trypsin (using trypsin to protein ratios of 1:10 or 1:50) by either incubating in aheated shaker (1 hour or ON at 37 °C) or by applying AFA in an R230. High confidence protein identifications are shown (A) as well
as a comparison of missed cleavages and peptide identifications between digestion methods (B).

Table 1. Adherent Cell Detachment
Workflow Comparison. A comparisonof AFA and trypsinization techniques
for the displacement of adherent cells
from 96-well polystyrene cell culture
plates shows the advantage of AFA.

R230 w/ AFA

Trypsinization

Wash Steps

23Pipetting Steps

48Centrifugation

N/A2xTime (min)1020 - 30

AutomatableYesNo

optimized for detachment (75 joules/10 scans) and subsequent homogenization(60 joules/10 scans) of the cell suspension directly in polystyrene culture plates (Figure 1).

A.

Number of Proteins Identifed: Adhesive Cell Culture

B.

Number of Peptides Identifed: Adhesive Cell Culture

1800

6000

1600

row treatment mode results in one step cell lysis, DNA shearing (reduction of viscosity), andprotein extraction. Extracted proteins are reduced and alkylated in the same plate, and thenbound to magnetic beads following Protein Aggregation Capture (PAC) conditions. Duringbead binding AFA (255 joules/10 scans) was applied. The bead-bound proteins were washedand resuspended in ammonium bicarbonate (ABC) buffer/trypsin solution. For hydrolysis,samples were incubated with intermittent AFA-mixing for 1 hour (90 joules/126 scans) at
34 °C. Released peptides were collected by harvesting the supernatant, which was finallytransferred to a new 96-well plate, dried using a speed vac, and reconstituted in buffer for
LC-MS analysis (Figure 2).
A.

B.

Figure 1. Displacement of Adherent Cells with AFA. Polystyrene cell culture plates with HCT 116 cells (80% confluent) are treatedwith low energy AFA settings to effect detachment of adherent cells in a single confluent cell-layer (A). A second AFA treatment
optimized for increased turbulence breaks apart the cell sheet into a cell suspension to facilitate transfer by pipetting into the 96
AFA-TUBE TPX 150 Plate (B).

1
Cell Culture
Polystyrene
Culture Plate

2

AFA

3

56

AFA

4

AFA

AFA

Mix
Suspension
or Detach
Adherent
Cells

Cell Lysisand DNA
Shearing

Reduction/Alkylation

BeadBinding

On-beadTrypsin
Digestion

Transfer to96 AFA-TUBE
TPX 150 Plate

Figure 2. AFA-enabled Workflow. AFA conditions for cell detachment, cell lysis, DNA shearing, and protein extraction weredeveloped with HCT 116 cells (Figure 1). AFA conditions for bead-binding and on-bead trypsin digest were developed using K562
protein extract (Figure 3).

Peptide Countfollowing addition of SDS (final concentration 1%). AFA treatment (13200 joules) in a row-by-Protein Count

Homogenized cell suspensions were transferred to a Covaris 96 AFA-TUBE TPX 150 Plate,

5000

1400
1200
1000
800600400CountC.

3000
2000
100020004000

Cell Detachment with AFA

Cell Detachment with Trypsin

1578

1663

Category Name (accession #)0

Cell Detachment with AFA

Cell Detachment with Trypsin

5108.255255.75

Series1
Count

Percent of protein category hits against total number
R230 - AFA (%)

Trypsinization (%)

extracellular matrix protein (PC00102)

0.3

0.6

cytoskeletal protein (PC00085)

7.4

7.0

transporter (PC00227)

5.3

5.3

scaffold/adaptor protein (PC00226)

3.4

3.5

DNA metabolism protein (PC00009)

1.6

1.8

cell adhesion molecule (PC00069)

0.7

0.8intercellular signal molecule (PC00207)

0.5

0.5

protein-binding activity modulator (PC00095)

4.4

4.7

viral or transposable element protein (PC00237)

0.3

0.2

RNA metabolism protein (PC00031)

10.3

9.5

calcium-binding protein (PC00060)

1.2

1.3

gene-specific transcriptional regulator (PC00264)

2.4

2.4

defense/immunity protein (PC00090)

0.9

0.8

translational protein (PC00263)

7.5

7.2

metabolite interconversion enzyme (PC00262)

13.9

13.5

protein modifying enzyme (PC00260)

8.0

8.7

chromatin/chromatin-binding, or -regulatory protein (PC00077)

3.2

3.3

transfer/carrier protein (PC00219)

0.7

1.0

membrane traffic protein (PC00150)

3.2

2.9

chaperone (PC00072)

3.1

3.1

cell junction protein (PC00070)

0.0

0.1

structural protein (PC00211)

0.2

0.2

storage protein (PC00210)

0.1

0.0

transmembrane signal receptor (PC00197)

1.1

1.4

Total Gene Hits in %

79.7

79.8

Figure 4. LC-MS Analysis of Tryptic Digestion of Protein Extract from HCT 116 Cells. A comparison of AFA and trypsin
detachment methods. After detachment of cells with either AFA (R230) or trypsinization, cells are transferred into a 96 AFA-TUBE
TPX 150 and lysed by applying AFA. The HCT 116 protein extract is then bound to magnetic beads using AFA-enhanced mixing,
followed by AFA-enhanced trypsin digestion. High confidence protein (A) and peptide (B) identifications are shown. PANTHER
analysis of protein categories classified by gene hits and LC-MS analysis of tryptic digestion of protein extract from HCT 116 cells(C) show similar protein ID coverage.

Discussion• Covaris AFA enables an automatable workflow starting from cell culture (adherent or suspension) to LC-MS ready protein hydrolysates.
• AFA conditions (energy, plate scanning or row-by-row treatment) are highly tunable and are employed throughout the workflow to accomplish four different functions.
• AFA simplifies adherent cell detachment by eliminating the need for cumbersome enzymatic incubation steps and improving user convenience.
• Following displacement of adherent cells, the entire workflow takes place in a Covaris 96 AFA-TUBE TPX 150 Plate, limiting transfer steps to a total of 3, and enabling the AFA treatments		

necessary for subsequent steps.

• Cell lysis and DNA shearing result from a single AFA treatment, extracting proteins and eliminating high viscosity due to high molecular weight nucleic acids.
• PAC workflow facilitates sample preparation by enabling compatibility with a wide range of buffer systems and simplifying cleanup.
• Immobilized protein (on-bead) trypsin digestion with AFA for 1 hour improves protein and peptide identifications over the standard overnight heat block incubation.
For any questions regarding this poster or its contents, please contact Ulrich Thomann (uthomann@covaris.com) or Patrick McCarthy (pmccarthy@covaris.com).
Information subject to change without notice. For research use only. Not for use in diagnostic procedures.
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