Biology subjects

Phlairaharn, T.

Publications and source records attributed to Phlairaharn, T..

3 recordsLinked to original sources

bioRxiv2023

Optimizing linear ion trap data independent acquisition towards single cell proteomics

A linear ion trap (LIT) is an affordable, robust mass spectrometer that proves fast scanning speed and high sensitivity, where its primary disadvantage is inferior mass accuracy compared to more commonly used time-of-flight (TOF) 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 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 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.

biochemistry↗

bioRxiv2022

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, mass spectrometry-based single-cell proteomics (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carried out comprehensive analysis of orbitrap-based data independent acquisition (DIA) for limited material proteomics. Notably, we found a fundamental difference between optimal DIA methods for high- and low-load samples. We further improved our low-input DIA method by relying on high-resolution MS1 quantification, thus more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we were able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we establish a complete experimental scp-MS workflow, combining DIA with accessible single-cell sample preparation and the latest chromatographic and computational advances and showcase our developments by profiling real single cells.

systems biology↗

bioRxiv2022

High sensitivity limited material proteomics empowered by data-independent acquisition on linear ion traps

In recent years, the concept of cell heterogeneity in biology has gained increasing attention, concomitant with a push towards technologies capable of resolving such biological complexity at the molecular level. For single-cell proteomics using Mass Spectrometry (scMS) and low-input proteomics experiments, the sensitivity of an orbitrap mass analyzer can sometimes be limiting. Therefore, low-input proteomics and scMS could benefit from linear ion traps, which provide faster scanning speeds and higher sensitivity than an orbitrap mass analyzer, however at the cost of resolution. We optimized an acquisition method that combines the orbitrap and linear ion trap, as implemented on a tribrid instrument, while taking advantage of the high-field asymmetric waveform ion mobility spectrometry (FAIMS) pro interface, with a prime focus on low-input applications. First, we compared the performance of orbitrap-versus linear ion trap mass analyzers. Subsequently, we optimized critical method parameters for low-input measurement by data-independent acquisition (DIA) on the linear ion trap mass analyzer. We conclude that linear ion traps mass analyzers combined with FAIMS and Whisper flow chromatography are well-tailored for low-input proteomics experiments, and can simultaneously increase the throughput and sensitivity of large-scale proteomics experiments where limited material is available, such as clinical samples and cellular sub-populations.

systems biology↗