Fall Quarter Overview

Grant Klausen Prell Lab

University of Oregon

Investigating Large Biological Complexes For Their Interactions and Structure

Using Native Mass For Investigating Biological Complexes

• Native mass spectrometry• High precision and

accuracy• Essentially unrestricted

mass range• Allows us to study both

mass and structure simultaneously

• Analyze multiple species in one spectrum

• Stoichiometry • Chemical identity

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Retaining Native Structure • ESI• Soft ionization technique

• Place protein of choice in specific volatile buffer• Spray it to retain its structure • Droplet of buffer evaporates• Kinetically trap protein

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Folded Unfolded

Our Instrument

Three S’s• Specificity • Selectivity• Sensitivity

Goal of the Lab: Studying Lipid Recruitment of Membrane Proteins Using Native Mass Spectrometry• Membrane proteins are

believed to associate with specific lipids• Potentially vital to assembly

and function of the protein• Determine native MS

techniques to study lipid recruitment• Other methods like X-ray

crystallography and Cryo-EM struggle with the heterogeneous nature of biolipids

Lipids have varying structures and charge properties• Zwitterionic• Negatively charged• Can adduct to basic sites

phosphocholine

phosphoethanolamine

cardiolipin

phosphoserine

sphingolipids

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Distinguishing Specific vs. Nonspecific Adduction

• Lipids readily adduct to proteins • In our case, non-membrane proteins and lipid head

groups

Easier to Unfold Protein than to Knock Adductions Off

Extent of Adduction for Acid Molecules Determined by Proton Affinity • Sodium and acid adduction are inversely related• Lower the charge state, more adductions seen • Less folded proteins result in more protonated

forms

Where We Are Headed• Effectively distinguishing between non-specific and

specific interactions• Testing full lipid adduction to non membrane

proteins • Develop approaches to prevent non-specific

adduction of lipids• Adding serine