3 research outputs found
Additional Precursor Purification in Isobaric Mass Tagging Experiments by Traveling Wave Ion Mobility Separation (TWIMS)
Despite
the increasing popularity of data-independent acquisition
workflows, data-dependent acquisition (DDA) is still the prevalent
method of LC–MS-based proteomics. DDA is the basis of isobaric
mass tagging technique, a powerful MS2 quantification strategy that
allows coanalysis of up to 10 proteomics samples. A well-documented
limitation of DDA, however, is precursor coselection, whereby a target
peptide is coisolated with other ions for fragmentation. Here, we
investigated if additional peptide purification by traveling wave
ion mobility separation (TWIMS) can reduce precursor contamination
using a mixture of <i>Saccharomyces cerevisiae</i> and HeLa
proteomes. In accordance with previous reports on FAIMS-Orbitrap instruments,
we find that TWIMS provides a remarkable improvement (on average 2.85
times) in the signal-to-noise ratio for sequence ions. We also report
that TWIMS reduces reporter ions contamination by around one-third
(to 14–15% contamination) and even further (to 6–9%)
when combined with a narrowed quadrupole isolation window. We discuss
challenges associated with applying TWIMS purification to isobaric
mass tagging experiments, including correlation between ion <i>m</i>/<i>z</i> and drift time, which means that coselected
peptides are expected to have similar mobility. We also demonstrate
that labeling results in peptides having more uniform <i>m</i>/<i>z</i> and drift time distributions than observed for
unlabeled peptides. Data are available via ProteomeXchange with identifier
PXD001047
A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test
Objectives Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. Methods The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. Results SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). Conclusions Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.</p
Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use
Objectives
During 2020, the UK’s Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity.
Methods
Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study.
Results
Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7–39.1) demonstrated diagnostic sensitivity of 92.4% (87.4–95.5), specificity of 97.4% (94.0–98.9) and near total concordance with RT-qPCR (Cohen’s Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1–99.3), specificity 97.4% (94.0–98.9) and Cohen’s Kappa 0.95.
Conclusions
This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.</p