Abstract:Targeted proteomics allows the highly
sensitive detection of specific
peptides and proteins in complex biological samples. Here, we describe
a methodology for targeted peptide quantification using a trapped
ion mobility quadrupole time-of-flight mass spectrometer (timsTOF
Pro). The prm-PASEF method exploits the multiplexing capability provided
by the trapped ion mobility separation, allowing more than 200 peptides
to be monitored over a 30 min liquid chromatography separation. Compared
to conventional parallel… Show more
“… Fig. 1 Timeline of selected milestones in the historic development of TIMS and PASEF acquisition modes depicted as a TIMS data structure with arbitrary peak heights ( 29 , 32 , 33 , 34 , 36 , 37 , 38 , 39 ) . PASEF, parallel accumulation–serial fragmentation; QTOF, quadrupole TOF; TIMS, trapped ion mobility spectrometry.…”
Section: Development Of Tims and Pasefmentioning
confidence: 99%
“…First demonstrated manually on a prototype instrument, online implementation required further development of the instrument electronics and firmware to handle the data in real time and to switch the quadrupole position rapidly on a submillisecond timescale. To date, and as discussed further later, the PASEF principle has been successfully implemented with data-dependent ( 29 ), targeted ( 38 ), as well as data-independent acquisition (DIA) ( 39 ) modes.…”
Recent advances in efficiency and ease of implementation have rekindled interest in ion mobility spectrometry, a technique that separates gas phase ions by their size and shape and that can be hybridized with conventional LC and MS. Here, we review the recent development of trapped ion mobility spectrometry (TIMS) coupled to TOF mass analysis. In particular, the parallel accumulation–serial fragmentation (PASEF) operation mode offers unique advantages in terms of sequencing speed and sensitivity. Its defining feature is that it synchronizes the release of ions from the TIMS device with the downstream selection of precursors for fragmentation in a TIMS quadrupole TOF configuration. As ions are compressed into narrow ion mobility peaks, the number of peptide fragment ion spectra obtained in data-dependent or targeted analyses can be increased by an order of magnitude without compromising sensitivity. Taking advantage of the correlation between ion mobility and mass, the PASEF principle also multiplies the efficiency of data-independent acquisition. This makes the technology well suited for rapid proteome profiling, an increasingly important attribute in clinical proteomics, as well as for ultrasensitive measurements down to single cells. The speed and accuracy of TIMS and PASEF also enable precise measurements of collisional cross section values at the scale of more than a million data points and the development of neural networks capable of predicting them based only on peptide sequences. Peptide collisional cross section values can differ for isobaric sequences or positional isomers of post-translational modifications. This additional information may be leveraged in real time to direct data acquisition or in postprocessing to increase confidence in peptide identifications. These developments make TIMS quadrupole TOF PASEF a powerful and expandable platform for proteomics and beyond.
“… Fig. 1 Timeline of selected milestones in the historic development of TIMS and PASEF acquisition modes depicted as a TIMS data structure with arbitrary peak heights ( 29 , 32 , 33 , 34 , 36 , 37 , 38 , 39 ) . PASEF, parallel accumulation–serial fragmentation; QTOF, quadrupole TOF; TIMS, trapped ion mobility spectrometry.…”
Section: Development Of Tims and Pasefmentioning
confidence: 99%
“…First demonstrated manually on a prototype instrument, online implementation required further development of the instrument electronics and firmware to handle the data in real time and to switch the quadrupole position rapidly on a submillisecond timescale. To date, and as discussed further later, the PASEF principle has been successfully implemented with data-dependent ( 29 ), targeted ( 38 ), as well as data-independent acquisition (DIA) ( 39 ) modes.…”
Recent advances in efficiency and ease of implementation have rekindled interest in ion mobility spectrometry, a technique that separates gas phase ions by their size and shape and that can be hybridized with conventional LC and MS. Here, we review the recent development of trapped ion mobility spectrometry (TIMS) coupled to TOF mass analysis. In particular, the parallel accumulation–serial fragmentation (PASEF) operation mode offers unique advantages in terms of sequencing speed and sensitivity. Its defining feature is that it synchronizes the release of ions from the TIMS device with the downstream selection of precursors for fragmentation in a TIMS quadrupole TOF configuration. As ions are compressed into narrow ion mobility peaks, the number of peptide fragment ion spectra obtained in data-dependent or targeted analyses can be increased by an order of magnitude without compromising sensitivity. Taking advantage of the correlation between ion mobility and mass, the PASEF principle also multiplies the efficiency of data-independent acquisition. This makes the technology well suited for rapid proteome profiling, an increasingly important attribute in clinical proteomics, as well as for ultrasensitive measurements down to single cells. The speed and accuracy of TIMS and PASEF also enable precise measurements of collisional cross section values at the scale of more than a million data points and the development of neural networks capable of predicting them based only on peptide sequences. Peptide collisional cross section values can differ for isobaric sequences or positional isomers of post-translational modifications. This additional information may be leveraged in real time to direct data acquisition or in postprocessing to increase confidence in peptide identifications. These developments make TIMS quadrupole TOF PASEF a powerful and expandable platform for proteomics and beyond.
“…The 4D label-free quantitative proteomic approach is highly sensitive and capable of accurate quantification (Meier et al, 2018;Lesur et al, 2021;Loginov et al, 2021). This approach can detect more low-abundance proteins, but many lowabundance proteins were not identified with western blotting (Kurien and Scofield, 2015).…”
The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.
“…Proteins separated by microchip electrophoresis can be trapped on the membrane by dragging the membrane which greatly reduces the separation and transfer time of Western blotting to a few minutes (within 1 hour) [101]. To make up for the shortcoming of the most methodologies and procedures used for the detection of glycomarkers, that is, too many experienced techniques and trained steps, in 2016, Lesur et al conducted an experiment in which the addition of lectin in the electrophoresis buffer was used in CGE to enable lectin affinity electrophoresis, and the liquid-phase binding assay facilitated the formation of the microfluidic-based automated immunoanalyzer [103]. In terms of dielectrophoresis, in 2011, Yang et al presented a high-throughput system assisted with dielectrophoresis for multiplexed detection of IL-2, IL-6, IL-10, and TNF-α.…”
Section: Electrophoresis Microfluidic System In Immunoassaysmentioning
In recent years, point-of-care testing has played an important role in immunoassay, biochemical analysis, and molecular diagnosis, especially in low-resource settings. Among various point-of-care-testing platforms, microfluidic chips have many outstanding advantages. Microfluidic chip applies the technology of miniaturizing conventional laboratory which enables the whole biochemical process including reagent loading, reaction, separation, and detection on the microchip. As a result, microfluidic platform has become a hotspot of research in the fields of food safety, health care, and environmental monitoring in the past few decades. Here, the state-of-the-art application of microfluidics in immunoassay in the past decade will be reviewed. According to different driving forces of fluid, microfluidic platform is divided into two parts: passive manipulation and active manipulation. In passive manipulation, we focus on the capillary-driven microfluidics, while in active manipulation, we introduce pressure microfluidics, centrifugal microfluidics, electric microfluidics, optofluidics, magnetic microfluidics, and digital microfluidics. Additionally, within the introduction of each platform, innovation of the methods used and their corresponding performance improvement will be discussed. Ultimately, the shortcomings of different platforms and approaches for improvement will be proposed.
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