Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics

Abstract

There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Herein, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.

Proteome-scale tissue mapping using mass spectrometry based on label-free and multiplexed workflows

“…We attributed the reduced measurement precision to low sensitivity of RTS-SPS-MS3, because ion loss could occur during multiple-level ion isolation and fragmentation process. 45…”

Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells

“…The field of single-cell proteomics (SCP) has advanced significantly in recent years regarding the proteome coverage of single cells due to the improvement of sample preparation, peptide separations, and mass spectrometry (MS). − Shifting from traditional bulk analysis to exploring the proteome at single cell levels offers valuable biological insights into the roles played by cell-to-cell heterogeneity in disease and development. − …”

Optimizing Linear Ion-Trap Data-Independent Acquisition toward Single-Cell Proteomics

“…Although DIA is typically performed on high-resolution mass spectrometers, historically, the method was first implemented on linear ion traps (LIT) . Compared to the OT, the LIT has proven to be a more sensitive and faster-scanning mass analyzer. , While the limited mass accuracy of LITs cannot make use of neutron-encoded isobaric tagging quantitative methods, LITs have sufficient resolution to quantify peptides using iTRAQ or 6-plex TMT in MS3 measurements, which Park et al demonstrated as can be applied to single cells . Additionally, LITs are capable of accurately quantifying fragment ions with low-input proteomics experiments using both parallel reaction monitoring and DIA. , However, all of these LIT-based low-input proteomics methods require either precursor ions to still be collected using a built-in OT or a spectrum library to be collected using an OT.…”

“…19,20 While the limited mass accuracy of LITs cannot make use of neutron-encoded isobaric tagging quantitative methods, 21 LITs have sufficient resolution to quantify peptides using iTRAQ or 6-plex TMT in MS3 measurements, 22 which Park et al demonstrated as can be applied to single cells. 23 accurately quantifying fragment ions with low-input proteomics experiments using both parallel reaction monitoring 24 and DIA. 25,26 However, all of these LIT-based low-input proteomics methods require either precursor ions to still be collected using a built-in OT or a spectrum library to be collected using an OT.…”