TY - JOUR

T1 - Optimizing Linear Ion-Trap Data-Independent Acquisition toward Single-Cell Proteomics

AU - Phlairaharn, Teeradon

AU - Ye, Zilu

AU - Krismer, Elena

AU - Pedersen, Anna Kathrine

AU - Pietzner, Maik

AU - Olsen, Jesper V.

AU - Schoof, Erwin M.

AU - Searle, Brian C.

N1 - Publisher Copyright: © 2023 American Chemical Society.

PY - 2023

Y1 - 2023

N2 - A linear ion trap (LIT) is an affordable, robust mass spectrometer that provides fast scanning speed and high sensitivity, where its primary disadvantage is inferior mass accuracy compared to more commonly used time-of-flight or orbitrap (OT) mass analyzers. Previous efforts to utilize the LIT for low-input proteomics analysis still rely on either built-in OTs for collecting precursor data or OT-based library generation. Here, we demonstrate the potential versatility of the LIT for low-input proteomics as a stand-alone mass analyzer for all mass spectrometry (MS) measurements, including library generation. To test this approach, we first optimized LIT data acquisition methods and performed library-free searches with and without entrapment peptides to evaluate both the detection and quantification accuracy. We then generated matrix-matched calibration curves to estimate the lower limit of quantification using only 10 ng of starting material. While LIT-MS1 measurements provided poor quantitative accuracy, LIT-MS2 measurements were quantitatively accurate down to 0.5 ng on the column. Finally, we optimized a suitable strategy for spectral library generation from low-input material, which we used to analyze single-cell samples by LIT-DIA using LIT-based libraries generated from as few as 40 cells.

AB - A linear ion trap (LIT) is an affordable, robust mass spectrometer that provides fast scanning speed and high sensitivity, where its primary disadvantage is inferior mass accuracy compared to more commonly used time-of-flight or orbitrap (OT) mass analyzers. Previous efforts to utilize the LIT for low-input proteomics analysis still rely on either built-in OTs for collecting precursor data or OT-based library generation. Here, we demonstrate the potential versatility of the LIT for low-input proteomics as a stand-alone mass analyzer for all mass spectrometry (MS) measurements, including library generation. To test this approach, we first optimized LIT data acquisition methods and performed library-free searches with and without entrapment peptides to evaluate both the detection and quantification accuracy. We then generated matrix-matched calibration curves to estimate the lower limit of quantification using only 10 ng of starting material. While LIT-MS1 measurements provided poor quantitative accuracy, LIT-MS2 measurements were quantitatively accurate down to 0.5 ng on the column. Finally, we optimized a suitable strategy for spectral library generation from low-input material, which we used to analyze single-cell samples by LIT-DIA using LIT-based libraries generated from as few as 40 cells.

U2 - 10.1021/acs.analchem.3c00842

DO - 10.1021/acs.analchem.3c00842

M3 - Journal article

C2 - 37338819

AN - SCOPUS:85164381392

VL - 95

SP - 9881

EP - 9891

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 26

ER -