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What is SWATH Mass Spectrometry?

The term proteome was first proposed by Australian scientists Williams and Wilkins in 1994. It refers to the sum of all proteins expressed by a cell or a tissue genome, and is the whole of all proteins corresponding to a genome.

In just 20 years, the research of proteome has made amazing progress, which is largely due to the application and continuous development of mass spectrometry technology in proteome. At the same time, the development of proteome and genome is complementary, and mass spectrometry can be used to identify the protein composition of any species whose genome has been sequenced. At present, the mainstream proteomics research program is the LC-MS/MS system. The LC-MS/MS system makes it possible to identify tens of thousands of proteins in one experiment.

The scheme based on LC-MS/MS system is also called shot-gun method, and the acquisition mode of mass spectrometry is DDA (Data Dependent Acquisition). In this mode, according to the intensity of the ionized peptide precursor ions, the mass spectrometer selects 10 to 20 ions with the highest intensity in sequence to enter the tandem mass spectrometer for further fragmentation, and then confirms by comparison with the template protein database. This method undoubtedly lost a lot of useful peptide precursor ion information.

In addition, after secondary fragmentation, only a small amount of spectra (about 30%) were resolved in the later identification using software, and most of the spectra were still not effectively used. Due to the randomness of the ions selected by mass spectrometry, the repeatability of identification is low, that is, the identification results of the same sample, the same instrument, and two different acquisitions are quite different. Due to the existence of space charge effect, the dynamic range of the analysis is greatly limited, and some important low-abundance proteins cannot be analyzed.

In response to the above problems, Dr. Ruedi Aebersold and his team of ETH Zurich and AB-SCIEX jointly launched a new mass spectrometry technology – SWATH (Sequential Windowed Acquisition of all Theoretical fragment ions).

SWATH acquisition mode is a new type of MS/MS scanning technology. It divides the scanning range into a series of intervals with 25 Dalton intervals, and obtains all fragment information of all ions in the scanning range through ultra-high-speed scanning. It is MS/MS -Extension of ALL technology. Taking the common scanning range of 400-1200 in the analysis of proteomics samples as an example, every 25 Dalton is used as a scanning interval (SWATH), and the scanning time of each SWATH is set to 100ms, then the scanning range needs a total of 32 SWATHs (1200-400). /25=32), it only takes 3.2 seconds to complete a scan.

Compared with the traditional shot-gun technology, the SWATH acquisition mode can scan all the peptide precursor ions in the scanning range at ultra-high speed and perform secondary fragmentation to obtain complete peptide information. Therefore, SWATH technology is a truly panoramic, high-throughput mass spectrometry technology. At the same time, it also solves the disadvantage of low repeatability of shot-gun identification.

In addition, with the help of the advanced Triple-TOF 5600 mass spectrometry system, SWATH has high accuracy and dynamic range in quantification. Different from traditional quantitative methods based on mass spectrometry, the quantitative method based on SWATH technology directly constructs the XIC of secondary fragment ions, and every point on the curve has sufficient mass spectral evidence, which greatly increases the accuracy and reproducibility of quantification.

The scientists used the SWATH acquisition mode to analyze the yeast cell whole protein lysate. The experimental results showed that the abundance of the detected proteins varied greatly, spanning 4 orders of magnitude. Moreover, the sensitivity and dynamic range of this method are comparable to the level of SRM analysis.

In 2010, AB-SCIEX launched the Triple-TOF 5600 system, which can “simultaneously” achieve high sensitivity, high resolution, mass accuracy and high mass spectrum acquisition speed on one instrument, and also has triple quadrupole mass spectrometry. level of quantitative ability.

Triple-TOF 5600 is a high-resolution mass spectrometer with powerful qualitative and quantitative capabilities developed by AB-SCIEX on the basis of the triple quadrupole mass spectrometer API 5500 and the quadrupole-time-of-flight mass spectrometer QSTAR Elite (QqTOF) system. The instrument, which successfully combines the latest technologies of triple quadrupole and time-of-flight mass spectrometry, is the first system to combine high accurate mass, high resolution, high scan rate and high sensitivity. The perfect combination of SWATH technology and TripleTOF 5600 makes SWATH technology play to the extreme.

The use of SWATH technology to study proteomics has already had many applications abroad. The Ruedi lab has published 4 papers related to the application of SWATH technology. Among them, the article published in the journal Nature method in September 2013 using affinity purification combined with SWATH technology (AP-SWATH MS to study protein interactions (Nature Methods 10, 1239–1245 (2013)) is very worthy of reference.

A major challenge of SWATH MS technology is the interpretation of complex mass spectral data. This is because in the SWAHT acquisition mode, all the fragment ions of all precursor ions in one window are presented in one spectrum, which cannot be effectively resolved using traditional protein identification software (such as mascot, etc.).

At present, a relatively reliable algorithm is generally recognized by the industry. This algorithm compares the retention time of the SWATH data, the shape of the fragment ion peak, the intensity of the fragment ion and other information with the corresponding information of the target spectral library and scores (subscore). A semi-supervised learning algorithm is used to iteratively train and classify all product ion peaks (traces) for each target peptide, and then linearly combine these subscores to obtain a total score value.

To evaluate the reliability of the results, the algorithm uses a DECOY idea similar to traditional protein database searches. At present, there are few software that use this algorithm to analyze SWATH data.