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Evaluating linear ion trap incorporation for MS3-based multiplexed single-cell proteomics

ABSTRACT: The project aims to evaluate the use of a linear ion trap to increase the sensitivity of multiplexed single-cell proteomics. Using diluted mouse peptide samples from C10 and Raw, we compared the proteome coverages, data missingness, and quantification performance between linear ion trap and Orbitrap acquisition modes for both MS2 and MS3 based analysis. The optimized RTS-LIT-MS3 method was further applied to the study of macrophage activation at single-cell resolution. Samples were labeled with 16-plex TMT. Data was searched with FragPipe.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Ying Zhu

PROVIDER: MSV000090178 | MassIVE | Mon Aug 22 14:15:00 BST 2022

REPOSITORIES: MassIVE

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Publications

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

Park Junho J Yu Fengchao F Fulcher James M JM Williams Sarah M SM Engbrecht Kristin K Moore Ronald J RJ Clair Geremy C GC Petyuk Vladislav V Nesvizhskii Alexey I AI Zhu Ying Y

Analytical chemistry 20230113

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 stud ...[more]

PMID: 36637389

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Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Project description:Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

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TKO6: A Peptide Standard to Assess Interference for Unit-Resolved Isobaric Labeling Platforms

Project description:Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from co-isolated and co-fragmented ions - commonly referred to as interference - remains a caveat of this technique. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely SPS-MS3) that can help alleviate interference. Yet, few standards are available to measure interference. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare the degree of interference in MS2 versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion trap-based TMT reporter ion analysis (IT-MS3). We show that the TKO6 standard is a valuable tool for assessing data quality and is particularly useful for benchmarking ion trap-based SPS-MS3 analyses.

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HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments

Project description:Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric tag-based quantification is “interference” which may be caused by co-eluting peptides that are co-isolated, co-fragmented, and co-analyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of TMTpro-labeled human peptides at a 1:1 ratio across all channels into which we spike TMTpro-labeled peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by 1) altering the MS2 isolation window width and 2) using different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run which can be compared across instruments to benchmark and track performance.

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