2 research outputs found
Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer
Liquid
chromatography (LC) prefractionation is often implemented
to increase proteomic coverage; however, while effective, this approach
is laborious, requires considerable sample amount, and can be cumbersome.
We describe how interfacing a recently described high-field asymmetric
waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray
ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer
(MS) enables the collection of single-shot proteomic data with comparable
depth to that of conventional two-dimensional LC approaches. This
next generation FAIMS device incorporates improved ion sampling at
the ESIāFAIMS interface, increased electric field strength,
and a helium-free ion transport gas. With fast internal compensation
voltage (CV) stepping (25 ms/transition), multiple unique gas-phase
fractions may be analyzed simultaneously over the course of an MS
analysis. We have comprehensively demonstrated how this device performs
for bottom-up proteomics experiments as well as characterized the
effects of peptide charge state, mass loading, analysis time, and
additional variables. We also offer recommendations for the number
of CVs and which CVs to use for different lengths of experiments.
Internal CV stepping experiments increase protein identifications
from a single-shot experiment to >8000, from over 100ā000
peptide
identifications in as little as 5 h. In single-shot 4 h label-free
quantitation (LFQ) experiments of a human cell line, we quantified
7818 proteins with FAIMS using intra-analysis CV switching compared
to 6809 without FAIMS. Single-shot FAIMS results also compare favorably
with LC fractionation experiments. A 6 h single-shot FAIMS experiment
generates 8007 protein identifications, while four fractions analyzed
for 1.5 h each produce 7776 protein identifications
Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening
Infrared matrix-assisted laser desorption electrospray
ionization
(IR-MALDESI) is a hybrid, ambient ionization source that combines
the advantages of electrospray ionization and matrix-assisted laser
desorption/ionization, making it a versatile tool for both high-throughput
screening (HTS) and mass spectrometry imaging (MSI) studies. To expand
the capabilities of the IR-MALDESI source, an entirely new architecture
was designed to overcome the key limitations of the previous source.
This next-generation (NextGen) IR-MALDESI source features a vertically
mounted IR-laser, a planar translation stage with computerized sample
height control, an aluminum enclosure, and a novel mass spectrometer
interface plate. The NextGen IR-MALDESI source has improved user-friendliness,
improved overall versatility, and can be coupled to numerous Orbitrap
mass spectrometers to accommodate more research laboratories. In this
work, we highlight the benefits of the NextGen IR-MALDESI source as
an improved platform for MSI and direct analysis. We also optimize
the NextGen MALDESI source component geometries to increase target
ion abundances over a wide m/z range.
Finally, documentation is provided for each NextGen IR-MALDESI part
so that it can be replicated and incorporated into any lab space