Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.
Elijah N McCoolJean M LodgeAbdul Rehman BasharatXiaowen LiuJoshua J CoonLiangliang SunPublished in: Journal of the American Society for Mass Spectrometry (2019)
Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.
Keyphrases
- artificial intelligence
- tandem mass spectrometry
- electron transfer
- mass spectrometry
- liquid chromatography
- ultra high performance liquid chromatography
- escherichia coli
- high performance liquid chromatography
- ms ms
- amino acid
- simultaneous determination
- high resolution
- sensitive detection
- gas chromatography
- big data
- induced apoptosis
- quantum dots
- solid phase extraction
- dna methylation
- squamous cell carcinoma
- small molecule
- protein protein
- staphylococcus aureus
- genome wide
- cell cycle arrest
- data analysis
- electronic health record
- density functional theory
- oxidative stress
- molecular dynamics
- label free
- transcription factor
- rectal cancer