Abstract
Here, we present native liquid extraction surface analysis (LESA) mass spectrometry imaging of proteins and protein complexes from mouse brain and liver tissue. Intact proteins were detected in characteristically low charge states, indicating that the proteins remain folded. In brain, abundant proteins such as ubiquitin and β thymosin 4 were detected homogeneously across the tissue whereas other proteins, such as neurogranin, were localised in specific anatomical regions. In liver, we demonstrate imaging of a protein complex (tetrameric hemoglobin), as well as fatty acid binding protein. Interestingly, the use of native-like solvents enables extraction of proteins which have not previously been observed in LESA experiments employing denaturing solvents, i.e., native LESA can be applied to extend the range of proteins observed. We also present native LESA ion mobility spectrometry and show that the collision cross sections of proteins extracted from tissue may be determined by travelling wave ion mobility spectrometry. The collision cross section of the 5+ ion of ubiquitin was calculated as 1047Å2, in good agreement with measurements of ubiquitin protein standard solutions. Collision cross sections for the 4+ ions of β-thymosin 4, β-thymosin 10 and two unidentified proteins were also calculated, together with that of a 10+ ion of an unidentified protein of molecular weight 15660Da.
Highlights
Native electrospray mass spectrometry (MS) has emerged as a powerful tool in the analysis of folded proteins and non-covalent protein complexes in the gas phase [1,2]
We present native liquid extraction surface analysis (LESA) ion mobility spectrometry and show that the collision cross sections of proteins extracted from tissue may be determined by travelling wave ion mobility spectrometry
A number of protein species detected in this experiment were not detected in previous LESA mass spectrometry imaging experiments performed with organic solvent systems, i.e., the results suggest that as well as maintaining tertiary structure, native LESA mass spectrometry increases the range of proteins detected
Summary
Native electrospray mass spectrometry (MS) has emerged as a powerful tool in the analysis of folded proteins and non-covalent protein complexes in the gas phase [1,2]. The growth of native mass spectrometry has been accompanied by the emergence of ion mobility spectrometry, which enables the shape and conformation of the folded protein or protein complex to be probed [6]. Classical (drift tube) ion mobility devices use a uniform electric field to drive ions through a cell pressurized with a background gas. The drift-times of ionic species facilitate the measurement, from kinetic theory, of an absolute collision cross-section ( ) [7,8]. Collision cross sections can be determined from TWIMS measurements by calibration against ions of known measured from drift tube measurements. Measurement of collision cross sections via TWIMS experiments allows derivation of information regarding molecular conformation [11,12,13]
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