Automation for Echo-MS with Access WorkstationFor large assays, Echo-MS

may be automated to save

manhours and facilitate

data collection overnight.

Automation is accomplished

by mating the Echo-MS

platform to Labcyte’s Access

workstation. In addition to the

robot arm for plate handling,

the workstation also manages

plate processing schema and

identification information via

the Tempo software platform.

Mass spec scan parameters

are still set independently by

the user.

Ion Transfer Interface

Waters Mass Spec Source384-well

Plate

Acoustic Transducer

High-Throughput Mass Spectrometry: Direct Acoustic Ionization for Sub-Second Sample ProcessingLucien Ghislain1, Martin Bachman2, Ian Sinclair2, Jonathan Wingfield2, Dan Addison2, Eric Hall1, Rick Stearns1, Lars Majlof1, Rich Ellson1, Rhys Jones3, Emmy Hoyes3, Steve Pringle3, Sammy Datwani1

1 Labcyte Inc. Sunnyvale, CA, USA; 2 AstraZeneca, Discovery Sciences, Alderley Park, Macclesfield, Cheshire, UK; 3 Waters, Wilmslow, UK;

© 2017 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

Presented at SLAS2017

POS-LGHT-1.0JAN 2017

FIGURE 1: Schematics of droplet production via standard ESI (left) and Echo-MS (right). In standard ESI, droplets are produced when a Taylor cone is formed at the end of a fluid capillary under a strong electric field. Using an acoustic transducer, droplets of similar dimensions may be produced directly from a microtitre plate well. When an electric field is applied during ejection, these droplets become charged.

FIGURE 4: Km determination (left) made from kinetic-mode data obtained in 30 minutes. Once assay conditions were optimized, the Echo-MS showed good concordance with LC-MS (right).

FIGURE 5: HTS data for 300,000-sample GSR inhibition assay. Final assay conditions for the HTS were 0.1 nM enzyme, 10 µM GSSG and 10 µM NADPH incubated for 60 minutes. Mass signal time traces of product and substrate (left) with a small section in focus (middle) to highlight active and inactive wells. When the product-substrate ratio is plotted on the plate map (bottom right), active wells are shown in dark blue.

FIGURE 8: Echo-MS Link network solution for data curation. The three system components of the Echo-MS platform are networked and coordinated together (top) to output both the MassLynx scan data as well as a text file annotating scan numbers with time stamps and plate and well ID, for use by GeneData Screener. The mass scan time traces are binned by well ID, using information provided by the Echo (bottom).

FIGURE 7: CAD drawing of an Echo-MS mated to an Access workstation for automation. The Access robot arm moves a plate from the stack and to the workstation’s barcode reader. The arm then moves the plate to the Echo-MS plate holder. Tempo software passes the barcode information to the Echo and initiates processing of the plate.

FIGURE 6: Two chemical mechanisms by which false positives are detected. An oxidative mechanism which returns glutathione back to the original substrate is shown on the left, while on the right is a compound that reacts with glutathione to remove it from the well

TABLE 1: Comparison of relevant parameters between standard ESI and the current breadboard implementation of Echo-MS. *Echo-MS sensitivity is constantly under improvement and measurement. Currently, the method is somewhere between two and three orders of magnitude less sensitive than standard ESI for a given analyte.

#1320

Electrospray Ionization Capillary Source

N2(g) Sheath Gas

Capillary OD 100 μm

600 kV/mElectric Field

200 kV/mElectric Field

Ultrasonic Fluid Ejection Cone

Charged Droplets(< 5 μm)

Charged Droplets(1 – 20 μm)

TaylorCone

Fluid Source(Capillary ID 50 μm)

Acoustic Echo-MS Source

FIGURE 2: A schematic of the latest ion transfer tube assembly, used for directing charged material from the 384-well microtitre plate to the mass spectrometer.

FIGURE 3: Breadboard implementation of the Echo-MS system for direct acoustic ionization.

Parameter ESI Echo-MS

Fluid Cone Base 10 – 50 μm 70 μm

Sample Flow 1 – 100 μL / min 5 μL / min

Droplet Size < 5 μm 1 – 20 μm

Electric Field 600 kV/m 200 kV/m

Inlet Temperature 350°C 200°C

Sensitivity femtomol sub-nanomol*

Introduction• We present progress toward commercial development of direct acoustic ionization mass

spectrometry for label-free, high-throughput screening (HTS).

• A customized Labcyte Echo acoustic liquid handler ejects charged droplets directly from a 384-well assay plate into a custom transfer interface. The resulting ions are detected by a Waters mass spectrometry (MS) instrument.

• A biochemical labelled assay has been modified to run on Echo-MS at speeds of 3 samples per second, eliminating the liquid chromatographic (LC) separation step and allowing for real-time assay development for label-free HTS.

• 300,000 samples were screened while operating in manual mode, and in concordance with HPLC-MS, to inform a cell-based drug-targeting workflow.

• Testing at scale demonstrated hardware robustness, as well as integration of the output with corporate data analysis tools to handle the big data generated.

• Instrument communication stamps spectra with sample ID information. Integration with robotic automation enabled 12 hours of walk-away operation.

Ion Generation with Echo-MSOur method for acoustic ion generation is conceptually similar to standard electro-spray ionization

(ESI) and may be discussed in analogous terms. In ESI, a fluid is pumped through a capillary and

subjected to an electric field, producing a Taylor cone which ejects charged droplets (FIG. 1, left). Our

method uses ultrasonic pulses delivered from an acoustic transducer to produce a fluid cone and a

spray of droplets (FIGURE 1, right). When an electric field is applied during ejection, those droplets

may be charged and desolvated to produce ions. Relevant comparison parameters between ESI and

our method are presented in TABLE 1.

This acoustic version of ESI, which we term Echo-MS, uses a modified Echo 555 acoustic dispensing

system to generate a mist of droplets in the femtolitre volume range directly from a standard 384-

well microtitre plate. These droplets pass through a charge field and produce ionized analytes as they

move through a transfer tube and are taken into the mass detector. Echo-MS can ionize a range of

small molecules, peptides, and proteins and can generate either +ve or –ve ions depending on the

polarity of the charge field.

G S R a s s a yE c h o M S 1 v s L C M S

% C o n v e rs io nE c h o M S

%

Co

nv

ers

ion

LC

MS

0 5 1 0 1 5 2 00

1 0

2 0

3 0

4 0

r2 = 0 .9 1

A g o n is ts

In a c t iv e s

In h ib ito rs

N A D P H K m

[N A D P H ] (u M )

v

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

0 .0

0 .5

1 .0

1 .5

2 .0

VmaxKm

1.47515.71

NADPH Km Echo-MS vs. LC-MS

Screening 300,000 SamplesA manually fed plate stage was used to screen over 600 plates of a glutathione-disulfide reductase

(GSR) inhibition assay in 10 working days. The assay was developed using a ‘kinetic mode’ on the Echo-

MS, including determination of assay Km in 30 minutes (FIG. 4, left). Re-engineered assay conditions

showed good concordance with LC-MS (FIG. 4, right). Improvements in sensitivity and process brought

the time taken to run a 384-well plate down from 9 minutes for the first 120 plates to 5 minutes for the

remainder of the screen. An excerpt of the data (FIG. 5, left) shows the ratio of substrate to product and

how this is linked back to compound plate maps to identify inhibitors of the enzyme (FIGURE 5, center).

Conclusions• Echo-MS uses direct acoustic ionization to deliver sub-second sample processing with MS.

• We have completed an acoustic MS assay to support an HTS against a biological target.

• Analysis of one 384-sample plate takes 5 minutes including all plate movements and data processing. Over 300,000 sample wells were analyzed in 10 working days .

• The assay hit rate was about 1% and actives were re-tested in a no-enzyme assay to remove false positives before being tested over a standard 12-point, half-log range.

• Reagent cost for a 1M compound screen was about $1500 compared to $150,000 for an equivalent, label-based HTS assay.

• Automation has been implemented on the system, allowing for continuous and overnight operation. A software platform has been developed to facilitate data curation and analysis.

Echo-MS Link Data Stamping for HTS Data CurationThe volume of data produced by the Echo-MS necessitates a robust data curation system for downstream

analysis. We have constructed a networked software solution between the Echo-MS system’s three

computer components to link mass scan data with plate and well information via the scan number. This

solution takes advantage of existing software (Labcyte’s Echo Server and Waters’ MassLynx) using a

custom program based on WREnS to collect real-time scan information. Upon completion of the assay,

the data is stitched together and analyzed via GeneData Screener.

False Positive MechanismsIf we remove the enzyme and substrate from the assay and just incubate the active compounds with

glutathione, we can discriminate those compounds that appeared active through a chemical rather than

biological mechanism (FIG. 6). Around 30% of the identified hits were shown to be false positives by

this approach.

Echo Mass Spec

Access Workstation

Echo-MS Storage

MassLynx PC

DataSync

MassLynx Data

Scan number Time-stampPlate and well ID

MassLynx

MS EPC

WREnS Server

Echo PC

Waters Client

Echo Server

A7A6A5A4A3A2A1

020406080

100120140160180200

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Coun

tsTh

ousa

nds

Time (s)