Page 1
High-throughput automation of LC-MS sample preparation: The SP3-iST workflow on the Freedom EVO® 100 and
the Resolvex® A200
2
¹Katrin Hartinger, ²Claudia Martelli, ³Katarzyna Buczak, ³Alexander Schmidt, 2Manuel Bauer and 1Nils A. Kulak
¹Katrin Hartinger, ²Claudia Martelli, ³Katarzyna Buczak, ³Alexander Schmidt,
¹PreOmics GmbH, Martinsried, Germany | ²Tecan Group Ltd, Mannedorf, Switzerland | ³Proteomics Core Facility, Biozentrum, University of Basel
¹PreOmics GmbH, Martinsried, Germany | ²Tecan Group Ltd, Mannedorf, Switzerland | ³Proteomics Core Facility, Biozentrum, University of Basel
Manuel Bauer and 1Nils A. Kulak
Automation of SP3-iST workflow
Automated solution for LC-MS sample preparation on Freedom EVO®
®
100 and Resolvex
Combination of versatile SP3 workflow and proven, high-quality iST
technology
Reproducible, standardized and high-throughput LC-MS sample
preparation
A200
A
3,000
2,500
2,000
1,500
# proteins
1,000
B
100
80
60
40
DiscussionResults
~3000 proteins IDs from
HEK293 cells
0
>=1
Excellent digestion efficiency
(< 10 % total missed cleavage)
Low technical variability
Materials & Methods
HEK293 cells
SP3 protein clean-up
iST peptide clean-up
LC-MS analysis
20
500
0
15 μg 25 μg
Automated
SP3-iST
Fig. 1| Performance of the automated SP3-iST workflow with different input amounts (6 replicates each) and the manual
SP3-iST workflow (3 replicates). High consistency in protein IDs (A) and percentage of missed cleavages (B).
50 μg 50 μg_m
Manual
SP3-iST
missed cleavage rate [%]
0
15 μg 25 μg
Automated
SP3-iST
50 μg 50 μg_m
Manual
SP3-iST
High consistency between
different protein input
amounts
Key features of workflow
PreOmics’ kits
and iST alkylation and
digestion on Freedom
EVO
®
100
on Resolvex® A200
Input: 15 µg, 25 µg or 50 µg of previously lysed HEK293 cells.
Workflow: Sample preparation was done employing the iST-PSI and the
SP3-iST Add-on kits (both PreOmics). Automated SP3 protein clean-up and iST
alkylation and digestion was implemented on the Freedom EVO® 100 (Tecan)
2.0
1.5
1.0
protein level
0.5
1.4e+10
1.0e+10
Intensity
High-throughput application:
96 samples within 5 hours
Highly automated: ~20
minutes hand-on time
Compatible with large variety
of lysis buffers
and automated iST peptide clean-up was performed on the Resolvex® A200
(Tecan; ´Automated SP3-iST´). As control for the automated protocol on the
Freedom EVO® 100, SP3 protein clean-up and iST alkylation and digestion was
performed manually in parallel with 50 µg protein input (´Manual SP3-iST´).
LC-MS analysis: Easy nLC 1000 coupled to QEplus instrument.
Data analysis: Progenesis QI software and MASCOT database search.
0.09
Coefficient of varience
0
15 μg 25 μg 50 μg 50 μg_m
Fig. 2| Technical variability of replicates for automated
SP3-iST workflow (6 replicates each) and for manual
SP3-iST workflow (3 replicates each).
0.12 0.11
Automated
SP3-iST
0.08
Manual
SP3-iST
2e+9
0 10
50 μg automated SP3-iST
50 μg manual SP3-iST
Fig. 3| High consistency in chromatograms for
automated SP3-iST and manual SP3-iST workflow with
50 µg input each.
20
30
40
50
60
70
80
Excellent sample quality
Ease-of-use & script-based
programming
www.preomics.com | [email protected]www.preomics.com | [email protected]
Loading...