Application advantages
Improve protein recognition and increase proteome coverage
Reliable identification even at very low concentrations
Perform LC / MS / MS analysis more efficiently to speed up the decision-making process
Introduction
As the complexity of bottom-up proteomics analysis continues to increase, it challenges the capabilities of mass spectrometers, which need to generate more detailed information to meet user needs. In recent years, the speed, sensitivity, and mass accuracy of the mass spectrometer have been significantly improved, resulting in better data quality, improved peptide sequence annotation, and more accurate identification results.
As hardware performance improves, we have developed a series of new LC / MS acquisition schemes and fragmentation mechanisms, including precursor ion discovery (PID) methods, information-independent acquisition (DIA), ion mobility (IM) assisted methods, and Electron transfer dissociation (ETD). At present, IM is mainly used for cross-sectional analysis and structural analysis of various types of analytes1, which can enhance the specificity of DIA acquisition, such as HDMSE. 2
This study demonstrates the advantages of a new high-definition data acquisition (HD-DDA) mode for protein and peptide identification, which incorporates ion mobility into a quadrupole time-of-flight mass spectrometer. HD-DDA adopts a high duty cycle mode, which is conducive to decision making and can realize experiments with high sensitivity and selectivity.
experiment
The cytoplasmic contents of E. coli and HeLa cells were digested with trypsin. The dissolved product was injected into the nanoACQUITY UPLC system equipped with an ACQUITY UPLC BEH 1.7 μm, 15 cm × 75 μm column and connected to a SYNAPT G2-Si mass spectrometer. Use ProteinLynx Global SERVER and / or Mascot to process and search the data3.
Results and discussion
HD-DDA improvements include full support for broadband enhancement4, which can increase the signal by 5 to 10 times, enhance decision-making logic, and switch between MS and MS / MS modes. Broadband enhancement uses ion mobility to separate product ions in the single charge state, combined with repeller synchronization to achieve a duty cycle close to 100%, as shown in Figure 1.
HD-DDA acquisition is usually performed in a non-targeting mode and can be supplemented by an unlimited target and / or exclusion list. The collision energy may be stepped, inclined, or determined in real time based on m / z and charge state. Data can be processed and searched through ProteinLynx Global SERVER or using vendor-neutral search algorithms and verification tools, such as Mascot and Scaffold5.
The advantages of broadband enhancement are confirmed in Figure 2. Among them, the E. coli trypsin digest was analyzed by nanoliter LC / MS / MC. Data collection uses conventional DDA and HD-DDA respectively. In both cases, 0.1 second MS / MS data at the same time in the LC peak was selected. In this experiment, after averaging the entire MS / MS spectrum, it was found that the signal was increased by 5 times.
In this experiment, 15 parallel MS / MS experiments were performed per survey scan. As shown in Figure 3, the results show that when comparing DDA with HD-DDA, the increase in MS / MS total ion current (TIC) is a function of the number of MS / MS channels. The average increase per run is 420%, which is consistent with the results shown in Figure 2. The inset is an example of 15 MS / MS channels, demonstrating that MS / MS data with good signal-to-noise ratio can be easily obtained from lower abundance peptides present in the sample.
Figure 4 shows that using HD-DDA to perform bottom-up LC / MS proteomics experiments on the same E. coli sample can increase sensitivity. Panel A shows the increase in the number of peptide sequence matches. The Wien intersection graph in Figure B compares the number of protein identifications. It can be observed that the number of identified peptides (34.8%) and protein (42.8%) have increased significantly.
Figure 5 shows the search results for a more challenging sample. The PLGS search results for the analysis of trypsin digests of HeLa cells are summarized here. In total, more than 2200 proteins were identified as exceeding 95% of the identification confidence threshold. In this experiment, the identification rate of the spectrum was 38%.
in conclusion
HD-DDA wideband enhancement usually enhances the signal by 5 to 10 times
The spectral data quality of low-abundance substances / peptides has been significantly improved
The ratio of MS / MS spectra to obtain correct matching is greatly increased
The increase in the number of HD-DDA data identifications is a direct result of increased sensitivity and improved spectral quality
references
1. Giles K. Travelling wave ion mobility. Int J Ion Mobility Spectrom.
2013; 16 (1): 1-3.
2. Rodriguez-Suarez E, Hughes C, Gethings L, Giles K, Wildgoose
J, Stapels M, Fadgen K, Geromanos SJ, Vissers JPC, Elortza F,
Langridge JI. An Ion Mobility Assisted Data Independent LC-MS
Strategy for the Analysis of Complex Biological Samples. Current
Anal Chem. 2013 April; 9 (2): 199-211.
3. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based
protein identi? cation by searching sequence databases using mass
spectrometry data. Electrophoresis. 1999 Dec; 20 (18): 3551-67.
4. Wildgoose, et. Al. A comparison of methods of improving the duty
cycle on orthogonal TOF mass analyzers. ASMS 2005.
4. Searle BC. Scaffold: a bioinformatic tool for validating MS / MS-based
proteomic studies. Proteomics. 2010 Mar; 10 (6): 1265-9.
Author: Keith Richardson1, Christopher J. Hughes1, Arkadiusz Grzyb1, Dominic Helm2, Bernhard Kuster2, Jason Wildgoose1
Unit: 1 Waters Corporation (Manchester, UK). 2 Munich University of Technology (Freising, Germany).
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