Orbitrap Exploris 480: The new generation of Q Exactive flagship (phase III)
In the first two issues, we introduced the design principles and hardware performance of the Orbitrap ExplorisTM 480, the superior qualitative capabilities, the optional components of the FAIMS Pro, and the improvements in TMT quantification. In this issue, Feifei will show you the best of Orbitrap ExplorisTM 480 –
Extremely rich quantitative process
BoxCar, SureQuant quantitative process
With the further development of proteomics, only Data Dependent Acquisition (DDA; such as non-labeled quantification, SILAC, TMT, stable isotope dimethyl labeling and other quantitative proteomics methods) are applicable to this stage. One category) has not been able to fully keep up with the ever-increasing analytical needs, especially for translational medical research in clinical cohorts. The subsequent development of DIA (1), PRM (2) and other targeted or quasi-targeting technologies further fill the gap between basic research and clinical applications.
The Orbitrap ExplorisTM 480 provides excellent support for all of the quantitative methods involved above, and the two new quantitative methods on the Orbitrap ExplorisTM 480 greatly enrich the existing quantification process.
In 2018, Matthias Labs published a new collection of BoxCars in Nature Methods (Figure 1).
Figure 1. BoxCar collection mode
The core idea of ​​the BoxCar acquisition method is to use segmentation accumulation in the first-level full scan, and the multi-plexing full scan of the combined scan to avoid the signal suppression of the low-abundance peptides in the same full scan, thereby enhancing one. The dynamic range of the full sweep and the sensitivity of the triggering level.
And with the Maxatch software's "Match Between Runs" feature, you can also use the exact mass-to-nuclear ratio and retention time of the first-order mass spectrometry feature to perform a quantitative mode that does not rely on secondary identification. BoxCar's acquisition mode has a significant advantage in identifying depth for samples with large dynamic range. The Orbitrap ExplorisTM 480 provides better compatibility with the Capture mode of the BoxCar, such as increasing the Spectral Multiplexing from 10 to 20 to support the more dense BoxCar isolation window. By analyzing plasma samples that have not been subjected to high-abundance protein removal, we can see that the collection mode using BoxCar significantly increases the number of peptides and proteins identified compared to conventional DDA (Fig. 2-(a,b,c) , d)).
Figure 2-(a,b,c,d) Analysis of plasma samples using the BoxCar DDA mode on the Orbitrap ExplorisTM 480
SureQuant-Ultra High Throughput PRM Quantification Method
Parallel reaction monitoring (Parallel Reaction Monitoring, PRM) on a high-resolution Orbitrap series instrument targeting quantitative methods, similar selective reaction monitoring (Selected Reaction Monitoring, SRM) on a triple quadrupole mass spectrometer, which is a potential marker for verification One of the gold standard methods.
However, PRM has been plagued by flux problems, and even with dynamic retention times, it is difficult to monitor hundreds of peptides simultaneously in a single shot. Therefore, in 2015, Gallien et al. published the Internal-Standard triggered PRM (IS-PRM) method on the MCP. The core idea is to perform a low resolution, low ion implantation time PRM scan of the internal standard peptide (internal standard The content is usually high, so it is not affected by the sensitivity), and then the real-time spectral search of the PRM scan is performed online, and if the target sequence can be matched, the high resolution of the endogenous peptide is triggered, and the PRM scan for the high ion implantation time is used. Quantitative (4) . This method has not been widely used due to the need to use the API provided by Thermo Fisher to program and modify the instrument control software.
The Orbitrap ExplorisTM 480 offers a commercial, ready-to-use SureQuant quantification method with a core idea similar to that of IS-PRM, but with a smarter and more convenient implementation. The SureQuant quantification process cleverly uses data-dependent acquisition methods to achieve the same functionality as IS-PRM (Figure 3).
Come see my new method
First, we will perform a first-level full scan, and then use the inclusion list triggered MS2 to scan the isotopically labeled internal standard peptides for secondary mass spectrometry (the inclusion list contains all the internal standard peptides to be detected). The target peptides are usually incorporated at higher concentrations, so the secondary mass spectrometry for the internal standard peptides will be performed with low resolution and low ion implantation time to save scan time as much as possible. We then performed a fast online spectral match on the secondary mass spectrum of the collected internal standard peptides, triggering a secondary mass spectrometry scan of the endogenous peptides if matched to the target internal peptide sequence (by precise mass) Shift to achieve), while the concentration of endogenous peptides is usually low, so we use a high-resolution, high-ion injection time secondary scan to maximize the sensitivity of the detection. In the SureQuant process, we do not set dynamic exclusion when performing secondary scans of DDA, so both the endogenous and internal standard peptides can obtain a complete PRM acquisition spectrum.
Figure 3. Schematic of the SureQuant quantitation process
for example
We use plasma protein quantification without high abundance protein removal as an example. The PQ 500 kit contains 500 heavy-isotope-labeled internal standard peptides of plasma proteins, for a total of 804 heavy-labeled peptides, which can be used for high-throughput targeting of plasma proteins. Using the SureQuant quantification process, we were able to quantify more than 550 endogenous peptides per needle by 70-minute gradient mass spectrometry, and the detection repeatability between the three needles was very high (Figure 4). The quantitative dynamic range ranges from 15 pmol to 4 amol on the column, spans greater than 6 orders of magnitude, and repeated injections exhibit a fairly high quantitative reproducibility (Figure 5).
Figure 4. Quantitative analysis of plasma samples without high-abundance protein removal using the SureQuant quantification procedure
Figure 5. Dynamic range and reproducibility of plasma protein quantification
New heights in proteomics
After three introductions, we found the Orbitrap ExplorisTM 480 mass spectrometer as the flagship model of the new generation Q Exactive series, a high-resolution mass spectrometer with high performance, stability and durability as the main considerations. It can meet the analytical needs of most proteomics, translational medicine and biopharmaceuticals, and significantly improve the quality and throughput of laboratory data. It is bound to take proteomics, especially clinical proteomics research to a new level.
references:
1. Bruderer, R., et al., Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Mol Cell Proteomics, 2015. 14(5): p. 1400- 10.
2. Peterson, AC, et al., Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88.
3. Meier, F., et al., BoxCar acquisition method enables single-shot proteomics at a depth of 10,000 proteins in 100 minutes. Nat Methods, 2018. 15(6): p. 440-448.
4.Gallien, S., SY Kim, and B. Domon, Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM). Mol Cell Proteomics, 2015. 14(6): p. 1630-44 .
Hospital intercom system,Medical intercom,Hospital call system
Zhuhai Mingke Electronics Technology Co., Ltd , https://www.zhmkdz-electronics.com