The Wu Lab | Top-down MS and Functional Proteomics

Detection of Protein-Protein Interactions using a 2-Dimensional Chemical Crosslinking Activity Correlated Proteomics
Platform (2D-XL-ACPP)
Morgan Mann | Hongyan Ma | Zhe Wang | Si Wu
Department of Chemistry & Biochemistry, University of Oklahoma, Norman, OK 73019

Overview 2D-ACPP XL-ACPP

Functional protein characterization remains one of the major challenges for Schematic of the 2D-ACPP
proteomics, owing in part, to the significant relationship between protein structure
and function. Current methods to determine higher order protein structure (i.e. X-

Normalized Intensity
Ray Crystallography, NMR, Pull-down etc.) are often difficult and time- Protein 3—Protein 5

consuming—requiring significant resources to isolate relatively few interactions. Protein 3—Protein n

To address this issue, we developed an ACPP that systematically correlates
proteins throughout a chromatographic separation to identify protein-protein Time

interactions. Furthermore, we applied a 2-Dimensional fractionation approach, Protein 1—Protein 2

• Some chromatographic separation methods can disrupt protein interactions and impact correlation.
which leveraged the good orthogonality between Size-Exclusion Chromatography
(SEC) and Anion Exchange Chromatography (AEX) to reduce random matching.

Normalized Intensity
Furthermore, we have begun implementing a Chemical Crosslinking (XL) step to Protein 2—Protein 3

facilitate the use of non-native separation methods with greater resolution.

Time

Hemoglobin 2-Dimensional 2-Dimensional

Background Mixture Fractionation
ACPP
Correlation
• Crosslinking agents —such as Disuccinimidyl suberate (DS))—can covalently link associated
• The 2D-ACPP can reduce the incidence of false-positives by requiring correlation in 2 Dimensions. proteins together to prevent dissociation.
• Proteins mediate almost all physiological and
pathological processes in organisms. • Developed and optimized using a commercially available mixture of Hemoglobin and blood proteins. • Crosslinking products can significantly add to sample complexity.
• e.g. Hemoglobin transport oxygen through
the blood.
Results Results
Chromatographic Orthogonality 2-Dimensional Correlation Crosslinking Efficiency Chromatographic Orthogonality
• Higher-order protein structure is determined
SEC XL AEX XL
by interactions with other proteins, which form SEC AEX SEC: R > 0.90
macromolecular complexes. AEX: R > 0.90
64 KDa
• Hemoglobin is a tetramer of two subunits.
• Protein function is determined by protein 32 KDa
structure. Figure 1. Structure of
Human hemoglobin. [2] 16 KDa
• Hemoglobin transports oxygen significantly Hemoglobin is composed of
more efficiently than monomeric two different protein subunits. Crosslinked
Hemoglobin
equivalents Hemoglobin
• Current methods to detect these interactions are difficult, slow, and require
significant amounts of sample preparation. Figure 4. SDS-PAGE Gel of the Figure 5. UV Chromatograms and SDS-PAGE Gels
Hemoglobin Mixture before and after
• Cannot be applied to biological samples in native-like conditions or helpful Figure 2. UV Chromatograms and SDS-PAGE Figure 3. Protein Interaction candidates
of the crosslinked sample during SEC & AEX
crosslinking. Crosslinked Hemoglobin does separation. Chromatogram peaks are shifted and
timeframes Gels of the Hemoglobin Mixture during both with comparison between separation not present a monomer (~16 KDa), and the broadened relative to the unlinked sample, which is
separation methods (SEC & AEX). Molecular methods. 9 Hemoglobin interactions were intensity of higher molecule bands is
• New, higher throughput methods will aid studies in a variety of fields, ranging consistent with increased sample complexity. SDS-
weight distributions indicate that the separation detected, out of 15 total interactions. 110 significantly increased—indicating that PAGE indicates that the separation methods are
from cancer and disease research to basic biochemistry. methods are orthogonal. proteins were detected and correlated in this presence of crosslinking products. orthogonal.
sample.

Protein ACPP Conclusions Future Directions

1. The ACPP can efficiently identify Hemoglobin proteins in a sample consisting of 1. Application to more complex samples to confirm efficacy in biological samples.
over 110 proteins. • E. coli lysate spiked with Hemoglobin (1:30).
2. The 2D-ACPP significantly reduced the number of non-Hemoglobin matches 2. Use of more powerful separation methods to evaluate the XL-ACPP.
Proteome
Size-Exclusion
Protein Co-Elution Statistical Correlation
detected. • Reverse-Phase Chromatography (RPLC).
Chromatography
3. Disuccinimidyl suberate can efficiently crosslink Hemoglobin. 3. In-vivo crosslinking to insure native conditions during crosslinking.
• Interacting proteins will coelute during separation and correlate well.
4. Size-Exclusion Chromatography and Anion Exchange Chromatography demonstrate 4. Improvements to the Correlation Algorithm
• A correlation coefficient (R-score) Threshold of 0.90 was utilized.
good orthogonality in both unlinked and crosslinked samples.
• Protein Interactions can be identified in a High-Throughput manner.
• Random Coelution can generate false-positives.

Contact Acknowledgements References

Morgan Mann, Undergraduate Research Assistant The authors would like to thank the members of the Wu Laboratory for their constant help and 1. Ma, H.; Delafield, D. G.; Wang, Z.; You, J.; Wu, S., Finding Biomass Degrading Enzymes Through an Activity-Correlated
morgan.w.mann@ou.edu guidance. The authors would also like to thank the University of Oklahoma for providing the Quantitative Proteomics Platform (ACPP). J. Am. Soc. Mass Spectrom. 2017, 28 (4), 655-663
101 Stephenson Parkway, Norman, OK 73019 opportunity to conduct and present this research 2. Zephyris. Hemoglobin. English language Wikipedia. https://commons.wikimedia.org/w/index.php?curid=2300973