Rapid quantitative analysis of fermentation broth samples toassess efficiency of engineered yeast strain turnoverUsing Acoustic Ejection Mass Spectrometry on the Echo® MS System
Rahul Baghla1, Chang Liu2, Rolf Kern1
1
SCIEX, USA, 2SCIEX, Canada
The field of synthetic biology has grown exponentially in recent
years due to advances in genetic engineering, microbiology,biochemistry and molecular biology among various otherdisciplines. The goal is to engineer microbes to create a desired
product; therefore the efficiency of production must be optimizedthrough both selection of proper environmental conditions andselection of the highest producing strain of the organism. Many
thousands of strains might need to be developed and assessed
before selection of one for scale up and use in commercial
production. This requires the quantitative analysis of the desired
product in many thousands of samples; and this needs to bedone as rapidly as possible to enable iterative strain refinement.
The SCIEX Echo MS System is a very high throughput,electrospray ionization mass spectrometry-based system thatuses Acoustic Droplet Ejection (ADE) to transfer a preciselycontrolled sample droplet from a well plate into the liquid streamof the Open Port Interface (OPI), which is directly coupled to theSCIEX Triple Quad™ 6500+ Mass Spectrometer. This analyticaltechnique has the sensitivity and compound coverage ofelectrospray mass spectrometry with the ability to analyze asample per second, in a non-contact, virtually carryover-freemanner. The small droplet size (2.5nL) provides a significant
dilution of matrix suppressing factors, which can be present incomplex samples, that helps to reduce preparation needed priorto analysis.
Here, the ability to detect a target compound directly out of yeast
fermentation broth, with minimal sample preparation, wasinvestigated to assess the utility of the Echo MS System for fast
screening of strains in a synthetic biology workflow.

Key features of Echo MS System for strain
selection• Samples were analyzed at 1 sec/sample, achieving highthroughput for screening large numbers of samples• Accurate quantification enabled differentiation between small
differences in concentration of product (Figure 1)• Minimal sample preparation was required to achieve therequired throughput and quantitative accuracy• Sensitivity of the Echo MS System provided high-quality dataat all concentrations tested

Figure 1. High reproducibility in quantification. The ability to
precisely quantify small differences in concentration of an analyte inbroth is critical during strain screening. Here, 10 replicates at eachconcentration of angiotensin in broth were analyzed out of the 1x
dilution sample. Statistically significant differences between the 25
and 30 mg/L samples were observed.
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Methods
Sample preparation: Yeast fermentation broth samples, spikedwith angiotensin (DRVYIHPFHL, MW=1295.6) were generously
supplied by a collaborator. The samples were prepared by lysing
the active broth with 4:1 acetonitrile/water and adding
angiotensin standard to represent 25, 30 and 50 mg/L
angiotensin in broth. The samples were then diluted to 40%
acetonitrile/60% water by volume to 1x, 10x and 100x,
centrifuged for 15 mins at 14000 rpm and transferred to a 96-wellplate for shipping. Samples were stored at -40 ºC until analysis.
Prior to analysis on the Echo MS System, 50 µL of each sample
was transferred to an Echo 384-well plate (Beckman Life
Sciences 384PP 2.0 Microplate).
Acoustic Ejection method: Development of an optimal acoustic
droplet ejection (ADE) method – the parameters involved ingenerating and transferring a sample droplet in the sample wellto the ESI source, involves a few parameters. First is selection ofan appropriate carrier solvent, analogous to the mobile phase inHPLC. 60:40 acetonitrile/water with 0.1% formic acid wasselected for this work, and the flow rate was optimized to 450µL/min.
Mass spectrometry: Echo MS System includes the SCIEXTriple Quad™ 6500+ LC-MS/MS System, and is controlled by
SCIEX OS Software. Angiotensin (DRVYIHPFHL) was monitored
using a single MRM transition (649.1 → 784.4 m/z, CE=40,
Dwell time = 30 msec). Source parameters for the OptiFlow® Ion
Source were GS1 of 90, GS2 of 70 and temperature of 600 ºC.
Data processing: Peak areas were integrated using SCIEX OSSoftware.

Table 1. Peak area and reproducibility results. 10 replicate ejections
were performed at each concentration and each dilution. Very good
reproducibility was observed across the dataset.
Concentration inbrothDilution# ofdroplets

Avg peakarea

Peak area%CV

25 mg/L

1x111174

5.230 mg/L

1x114787

7.950 mg/L

1x131078

5.5

25 mg/L

10x461064.230 mg/L

10x47269

7.5

50 mg/L

10x

416202

5.2

25 mg/L

100x

101386

14.630 mg/L

100x

1016645.750 mg/L

100x102982

7.5Accuracy of quantification
Samples were provided as a series of three 10x (1x, 10x and100x) dilutions of three concentrations of angiotensin in broth(25, 30 and 50 mg/L). Samples were analyzed in replicates of 10
to show reproducibility of the AEMS technique in this matrix(Figure 2). Very high reproducibility was observed across alldilutions, even the least dilute sample (1x), indicating thatminimal sample preparation could be used in this type ofexperiment to achieve desired results (Table 1). In addition, thereproducibility was high enough to easily distinguish between thesame concentration differences used here (Figure 1), which is
key when making critical decisions during strain selection.

Figure 2. Raw MS data for angiotensin peptide in undiluted 1x sample. Ten replicates were analyzed for each concentration, acquired with a single
droplet ejection method. Very high reproducibility was achieved for all 3 concentrations with minimal preparation of the sample (Table 1).

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Linearity and precision is shown for the three dilution series inFigure 3. As a sample is more diluted, analyte signal can beincreased by increasing the droplet count for sample ejection.
For the 10x diluted samples, a 4 droplet method was used toachieve good results. For the 100x diluted samples, a 10 droplet
method was used, also with good results, although the lowest
point for that series is showing higher variability than might bedesired. But in all 3 dilutions, good linearity was observed.

Conclusions
The results of this study show that the Echo MS System iscapable of rapidly producing high-quality results for the analysis
of a target peptide in a minimally prepared, commonfermentation broth matrix. The rapid analysis time of this platform
will enable faster generation of quantitative data to assess
microbe strain efficiency, which should allow for more rapid
strain selection removing a costly bottleneck in the important andgrowing field of synthetic biology.

References
1.

Rapid MS/MS analysis with Acoustic Ejection Mass
Spectrometry (AEMS) - Using the SCIEX Echo® MS System
to break bottlenecks in quantitative mass spectrometry
throughput. SCIEX technical note RUO-MKT-02-11385-A.

Figure 3. Concentration curve for angiotensin in all three brothdilutions. Good linearity and reproducibility were observed for thesample concentrations out of the minimally prepared samples.

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