Microanalysis of Brain Angiotensin Peptides Using Ultrasensitive Capillary Electrophoresis Trapped Ion Mobility Mass Spectrometry

Abstract

While the role of the renin–angiotensin system (RAS) in peripheral circulation is well characterized, we still lack an in-depth understanding of its role within the brain. This knowledge gap is sustained by lacking technologies for trace-level angiotensin detection throughout tissues, such as the brain. To provide a bridging solution, we enhanced capillary electrophoresis (CE) nanoflow electrospray ionization (ESI) with large-volume sample stacking and employed trapped ion mobility time-of-flight (timsTOF) tandem HRMS detection. A dynamic pH junction helped stack approximately 10 times more of the sample than optimal using the field-amplified reference. In conjunction, the efficiency of ion generation was maximized by a cone-jet nanospray on a low sheath-flow tapered-tip nano-electrospray emitter. The platform provided additional peptide-dependent information, the collision cross section, to filter chemical noise and improve sequence identification and detection limits. The lower limit of detection reached sub-picomolar or ∼30 zmol (∼18,000 copies) level. All nine targeted angiotensin peptides in mouse tissue samples were detectable and quantifiable from the paraventricular nucleus (PVN) of the hypothalamus even after removal of circulatory blood components (perfusion). We anticipate CE-ESI with timsTOF HRMS to be broadly applicable for the ultrasensitive detection of brain peptidomes in pursuit of a better understanding of the brain.

Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments

“…A number of commercial nanoLC systems and columns provide a reasonable combination of sensitivity and efficiency for single-cell proteomics, and these are recommended for most practitioners. Alternative high-resolution separation techniques employing orthogonal separation mechanisms, for example, capillary electrophoresis and ion mobility, as well as multidimensional techniques may potentially be employed as front-end approaches in MS-based single-cell proteomics 11,46 . Increasing ion transmission in the mass spectrometer is generally the purview of instrument developers and companies, and future gains in this area are expected to further benefit single-cell proteomics.…”

Developments in Trapped Ion Mobility Mass Spectrometry to Probe the Early Stages of Peptide Aggregation

“…Ion Mobility Mass Spectrometry (IM-MS) has proven to be a powerful method for the analysis of m / z selected ions within a heterogeneous mixture of low concentrated analytes. With IM-MS, the mobility of charged molecules is measured, allowing the separation of these ions based on their size, mass, and charge, which is related to the overall 3-dimensional structure of the ion. − For example, the addition of ion mobility to mass spectrometry allows one to separate and identify isomeric compounds − or to unravel the conformational landscape of biomolecular assemblies. − These IM-MS capabilities make it an ideal method to unravel the pathways of amyloid formation and to obtain oligomeric separation between charge states with similar m / z value. As of today, several ion mobility-based approaches have been developed, and the most common ones are Drift Tube (DTIMS), Differential (DIMS), Field Asymmetric (FAIMS), Traveling Wave (TWIMS), and more recently, the focus of this work, Trapped Ion Mobility Spectrometry (TIMS). ,− …”

Electrochemically Etched Tapered-Tip Stainless-Steel Electrospray-Ionization Emitters for Capillary Electrophoresis–Mass Spectrometry

“…Electrospray-ionization emitter tips used in mass spectrometry are usually made by blunt-end cutting either fused-silica or stainless-steel tubes; or by pulling a tubular resin under heat to produce a fine tip, such as borosilicate glass, or fused silica. − In addition to these types of emitters, tapered-tip emitters have been deemed very useful for electrospray ionization for approaches to achieve interfacing of capillary electrophoresis to mass spectrometry. − While pulled emitters are routinely made with borosilicate glass and fused-silica capillaries, this technique is not suitable for making stainless-steel emitters for electrospray ionization. For applications that require stainless-steel emitters, production of these are limited to mechanical cutting that also involve deburring and grinding down any roughness of the surface to achieve a straight and blunt tip.…”

“…8,10 While tapered-tip fused-silica capillaries are very useful for a wide range of applications, the use of tapered-tip stainless-steel emitters have recently been shown to be of great benefit for sheath-flow assisted electrospray ionization for connection of capillary electrophoresis to mass spectrometry, providing up to 100-fold improved sensitivity when compared to blunt tip emitters. 11,12 Here, we present how a tapered stainless-steel emitter may be produced with the use of materials and chemicals available as household products: oxalic acid and a USB power adapter. The electrochemical reaction used here for production of electrospray-ionization emitters is turned on, is left for ∼1 h and eventually the etching process self-inhibits, producing symmetrical tapered tips and removing the need for manual grinding and deburring of the emitter.…”