Data-Dependent Acquisition Ladder for Capillary Electrophoresis Mass Spectrometry-Based Ultrasensitive (Neuro)Proteomics

Abstract

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in sensitivity in high-resolution mass spectrometry (HRMS). Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. Here, we systematically evaluate performance limitations in this technology to develop a data acquisition strategy with deeper coverage of the neuroproteome from trace amounts of starting materials than traditional dynamic exclusion. During standard data-dependent acquisition (DDA), compact migration challenged the duty cycle of second-stage transitions and redundant targeting of abundant peptide signals lowered their identification success rate. DDA was programmed to progressively exclude a static set of high-intensity peptide signals throughout replicate measurements, essentially forming rungs of a "DDA ladder." The method was tested for ∼500 pg portions of a protein digest from cultured hippocampal (primary) neurons (mouse), which estimated the total amount of protein from a single neuron. The analysis of ∼5 ng of protein digest over all replicates, approximating ∼10 neurons, identified 428 nonredundant proteins (415 quantified), an ∼35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder enhances CE-HRMS sensitivity to single-neuron equivalent amounts of proteins, thus expanding the analytical toolbox of neuroscience.

Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

“…The neuron samples were analyzed on a microanalytical CE-ESI-HRMS system that we custom-built and validated for ultrahigh sensitivity following our recent studies. ,, Peptides from the somal protein digests were electrophoresed in 25% acetonitrile (ACN) with 1 M formic acid (background electrolyte) and then ionized in a coaxial low-flow sheath-flow CE-ESI interface. This CE-ESI platform was constructed based on an independent design, , which we operated in the cone-jet spraying regime to maximize detection sensitivity. , Peptide ions were detected on a hybrid quadrupole orbitrap mass spectrometer, equipped with a higher-energy collisional dissociation cell. Tandem MS was controlled by a data-dependent acquisition executing dynamic exclusion.…”

“…These performance metrics make CE–HRMS proteomics attractive for integration with patch-clamp electrophysiology. Recently, we custom-built CE–HRMS platforms with a capability for ∼250 zmol lower limit of detection for model peptides. , These instruments supported the identification of ∼200–800 proteins in protein digests estimating to ∼5–10 neurons. ,, A data-dependent acquisition ladder improved the duty cycle economy, identifying 428 proteins from single-cell equivalent protein amounts from cultured neurons . We recently explored the technical possibility to combine electrophysiology with single-neuron CE–HRMS proteomics. − …”

“…35,36 These instruments supported the identification of ∼200−800 proteins in protein digests estimating to ∼5−10 neurons. 35,37,38 A data-dependent acquisition ladder improved the duty cycle economy, identifying 428 proteins from singlecell equivalent protein amounts from cultured neurons. 38 We recently explored the technical possibility to combine electrophysiology with single-neuron CE−HRMS proteomics.…”

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

“…21,22 Chemical and spectral interferences were, however, found to challenge peptide identification and quantification, especially on low-abundance signals that are common to trace-level analyses. 23,24 Gas-phase separation via ion mobility HRMS offers remedy without risking loss or dilution to the sample. 25 As the collision cross section (CCS) is an intrinsic property of peptides and CCS can be derived from measured ion mobility, ion mobility HRMS enhances the fidelity of identifications with another dimension of a compound-dependent property, the CCS.…”

“…HRMS detection is rapid, can be made quantitative, and does not require the use of functional probes or chemical labeling. Recent developments in HRMS achieved sub-attomole lower limits of detection (LLOD) for peptides from limited sample amounts. , Chemical and spectral interferences were, however, found to challenge peptide identification and quantification, especially on low-abundance signals that are common to trace-level analyses. , …”