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Emerging Biomedical Analysis
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
Q-TOFMS and Q-OTMS are operated with similar principles. The quadrupole can be used as a path to transfer all of the ions or as a mass filter to select specific ions. The TOF or Orbitrap serves as a high-resolution mass analyzer for accurate mass measurement. In these types of instruments, data-dependent acquisition (DDA) and data-independent acquisition (DIA) are the two commonly used acquisition modes. In DDA mode, a high-resolution survey scan of all precursor ions is first acquired in the TOF or Orbitrap. Several MS/MS scans of the selected ions are then acquired based on the survey scan according to a certain rule, typically on ions exhibiting the highest intensities in the survey scan. DDA allows the data acquisition of certain ions without any information prior to the analysis. It is very useful for large-scale unknown sample analysis when combined with front-end separation techniques, such as HPLC or CE. DIA involves data acquisition within a predefined mass range. SRM is one of the DIA strategies used for quantitatively measuring samples with known information. Other DIA strategies, such as MSE and sequential window acquisition of all theoretical fragment ion spectra (SWATHTM), can be applied to analyze large-scale protein mixtures.
Detection of Food Allergen Residues by Immunoassays and Mass Spectrometry
Published in Andreas L. Lopata, Food Allergy, 2017
Sridevi Muralidharan, Yiqing Zhao, Steve L. Taylor, Nanju A. Lee
The shotgun proteomics workflow is suited for discovery studies and typically adopts the bottom-up approach, where proteins are pre-fractionated, subjected to proteolytic digestion, followed by nanoflow-liquid chromatography- tandem mass spectrometry (nLC-MS/MS). This workflow is becoming ideal for identifying proteins and characterizing post translational modifications. Alternatively, top-down approach is more suited for characterizing intact proteins typically analysed by high resolution mass analysers, followed by MS/MS based on collision induced dissociation of ions. Gene ontology annotations and analysis have been possible from using databases such as UniProt, Interpro, KEGG, and new tools such as WEGO (Ye et al. 2006), MaxQuant (Cox and Mann 2008), STRAP (Bhatia et al. 2009), DAVID or MAPMAN. This approach is predominantly used for biomarker discovery across different areas of research but has recently been successfully demonstrated for discovery of potential allergens in uncharacterised novel allergenic foods (in house unpublished). As an alternative, data acquired using data-independent acquisition mode based on simultaneous fragmentation activation of all ions (all co-eluting peptides) could exponentially enhance the depth of data available for analysis, and has the potential for multiple retrospective analyses from a single experiment. This approach has been exploited in certain other areas of research, however it requires the generation of high quality spectral libraries for peptide identification and quantification and its application in food allergen research is still at infancy.
Metabolomics and Proteomics
Published in Crystal D. Karakochuk, Kyly C. Whitfield, Tim J. Green, Klaus Kraemer, The Biology of the First 1,000 Days, 2017
Richard D. Semba, Marta Gonzalez-Freire
There are three general approaches that are used in mass spectrometry-based proteomics: (1) data-dependent acquisition (DDA) usually done using Orbitrap mass analyzers; (2) targeted proteomics using SRM; and (3) data independent acquisition (DIA) using SWATH [21] (Figure 30.1). Samples are typically prepared using extraction and digestion with an enzyme such as trypsin. Large epidemiological studies can utilize a 96-well plate format and robotics for high throughput and lower variability in processing [22]. In DDA, a full spectrum of peptides is required of all the ion species that coelute at a certain point during the gradient and then fragmented at the MS1 level. This is followed by the collection of fragmentation spectra at the MS2 level that are used to identify the peptides. The instrument alternates between MS1 and MS2, but between cycles the ion species that are not fragmented in MS1 are lost and not detected. The Orbitraps are currently limited to a capacity of ~1 million ions with a restricted dynamic range. Targeted proteomics using SRM is based upon the fragmentation of peptides based upon their known mass-to-charge ratio (m/z) in the first quadrupole, followed by the collection of fragmentation spectra in the third quadrupole. Recently, as reported by Kusebauch and colleagues, it has theoretically been possible to detect and quantify any of the 20,277 proteins of the protein-coding genes in the human proteome using SRM [23]. The Human SRMAtlas currently consists of 166,174 proteotypic peptides representing the human proteome [23]. SRM assays can be multiplexed to simultaneously detect and quantify large sets of proteins in plasma [24]. Conventional antibody- or aptamer-based approaches that rely upon the recognition of epitopes often cannot differentiate highly homologous proteins or protein variants based upon a single amino acid substitution, and these limitations can be overcome by use of SRM [25]. In DIA, all peptides are fragmented continuously across the entire mass range, giving rise to large multiplexed spectra. The dynamic range of DIA is about 4 to 5 orders of magnitude. There are now SWATH-MS libraries that can facilitate the identification of >10,000 plasma proteins [26]. One advantage of using SWATH is that once SWATH-MS data have been collected in an experiment, the data can be interrogated again in the future, as SWATH-MS libraries continue to evolve in their coverage.
Advances in phosphoproteomics and its application to COPD
Published in Expert Review of Proteomics, 2022
Xiaoyin Zeng, Yanting Lan, Jing Xiao, Longbo Hu, Long Tan, Mengdi Liang, Xufei Wang, Shaohua Lu, Tao Peng, Fei Long
The two main modes of mass spectrometry for the analysis of complex protein mixtures are ‘top-down’ and ‘bottom-up’ separation modes. Top-down refers to separating the mixture at the protein level, followed by enzymatic digestion and mass spectrometry. In contrast, ‘bottom-up’ refers to the enzymatic digestion of the extracted total protein into a mixture of peptides, followed by separation and mass spectrometry [89,90]. Mass spectrometry-based bottom-up experiments can be divided into data-dependent acquisition (DDA) and data-independent acquisition (DIA) [91]. DDA scans the precursors and selects the most abundant ions for fragmentation and secondary mass spectrometry in the primary scan. However, the random nature of this method makes phosphorylated proteomics poorly reproducible. On the other hand, DIA makes up for the shortcomings of DDA by fragmenting all precursors for detection, which dramatically improves the coverage and reproducibility of the peptides. Nevertheless, the spectra generated using this method are too complex and challenging to analyze. Researchers commonly use DDA deep library building to assist the DIA data analysis method [92].
Unraveling the complexity of the extracellular vesicle landscape with advanced proteomics
Published in Expert Review of Proteomics, 2022
Julia Morales-Sanfrutos, Javier Munoz
The standard method for collecting MS spectra is data-dependent acquisition (DDA). Despite its simplicity, DDA presents some caveats, mainly related to the stochastic nature of peptide fragmentation, which results in high rates of missing values. This is particularly problematic in large longitudinal studies and when sample amount is limited, a typical scenario in clinical studies. The recent development of Data-Independent Acquisition (DIA) modes overcomes the inherent irreproducibility and under-sampling of DDA. DIA acquires MS/MS spectra for, theoretically, all peptide precursor ions and it does so without bias to precursor ion selection. Not surprisingly, several authors have begun to exploit the potential of DIA approaches for the analysis of sEVs [65–68]. A crucial parameter in proteomics is the dynamic range, which is the ability to detect low abundance proteins in the presence of highly abundant ones. A way of improving the dynamic range is by means of gas-phase separation using high-field asymmetric waveform ion mobility spectrometry (FAIMS) in Orbitrap-based instruments [69] or even the combination of trapped ion mobility with time-of-flight MS [70, 71], both of which have shown much promise for proteomic applications.(3) Computational approaches
New advances in quantitative proteomics research and current applications in asthma
Published in Expert Review of Proteomics, 2021
Yanting Lan, Xiaoyin Zeng, Jing Xiao, Longbo Hu, Long Tan, Mengdi Liang, Xufei Wang, Shaohua Lu, Fei Long, Tao Peng
To address this issue, an alternative method by utilizing data-independent acquisition (DIA) has been developed. The DIA technique does not require cycling between MS1 and MS2 modes for peptide identification. Instead, it allows rapid switching of collision energies between low and high energy levels while providing information on the precursor and product ions of all isotopes within the entire chromatographic peak width [1,57,58]. As a result, the intensities of all ions detected in the MS1 scan are available for quantification, which improves the reliability of abundance estimates. However, more sophisticated software is required to reassociate precursor and product ions and to identify and match peptide spectra throughout the experiment based on accurate mass and retention times (AMRT) [58]. In 2015, Bruderer et al. first reported and detailed hyper reaction monitoring (HRM), a novel DIA mass spectrometry technique. The researchers found that the reproducibility of HRM in peptide detection was above 98%, resulting in quasi-complete data sets compared with only 49% of shotgun proteomics [59]. Therefore, the most obvious and direct advantage of the HRM approach for multiple sample analysis is that a quasi-complete data matrix can be obtained without any interval comparison. In addition, missing data is the greatest limitation of shotgun proteomic for quantification [60]. The HRM approach fundamentally solves this limitation. In conclusion, using DIA to obtain high content discovery is a novel approach, which can be used as the preferred approach for quantitative analysis of proteins in multiple samples.