2022
DOI: 10.1021/acs.analchem.1c02929
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Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples

Abstract: In this work, we developed an ultra-sensitive CE-MS/ MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-ofthe-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultrasensitive CE-MS… Show more

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Cited by 29 publications
(25 citation statements)
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“…35 Due to the biological significance of PTMs, it is necessary to have highly sensitive proteomic methods that can detect these low abundance modified peptides. We have demonstrated an advantage previously in CE-MS/MS for profiling these modified peptides without the need for any prior enrichment, 17 and FAIMS has been reported to improve identification of phosphorylated peptides due primarily to the increased signal-to-noise ratio. 20 For the optimal CE-FAIMS-MS/MS method, 76 ± 9 unique phosphopeptides were identified from 1 ng of the HeLa protein digest standard, representing an ∼46% increase over CE-MS/MS alone (Figure 5A).…”
Section: Effect Of CV In Faims Experiments On Identification Of Post-...mentioning
confidence: 98%
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“…35 Due to the biological significance of PTMs, it is necessary to have highly sensitive proteomic methods that can detect these low abundance modified peptides. We have demonstrated an advantage previously in CE-MS/MS for profiling these modified peptides without the need for any prior enrichment, 17 and FAIMS has been reported to improve identification of phosphorylated peptides due primarily to the increased signal-to-noise ratio. 20 For the optimal CE-FAIMS-MS/MS method, 76 ± 9 unique phosphopeptides were identified from 1 ng of the HeLa protein digest standard, representing an ∼46% increase over CE-MS/MS alone (Figure 5A).…”
Section: Effect Of CV In Faims Experiments On Identification Of Post-...mentioning
confidence: 98%
“…In this work, we evaluated coupling the FAIMS Pro interface with ultrasensitive sheathless CE-MS/MS 17 for proteomic profiling of low nanogram samples. The ultrasensitive CE-MS/ MS method used here was optimized to produce a minimal electroosmotic flow (EOF), thereby resulting in a wider separation window than typically observed with bare fused silica (BFS) capillaries (∼60 min).…”
Section: Coupling Ce-ms/ms With Faims and Optimization Of Ce-faims-ms...mentioning
confidence: 99%
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“…Microanalytical platforms were built for CE to enable handling nanoliters of samples. Low sheath-flow CE-ESI interfaces capable of sub-attomole-to-zeptomole detection were hyphenated to quadrupole Orbitrap, time-of-flight (TOF), , and quadrupole-orbitrap-ion trap tribrid , mass spectrometers for detection. The technology was able to quantify the RAS peptides in microscale biopsies (punches) from (control) PVN in ca.…”
Section: Introductionmentioning
confidence: 99%
“…IEX typically uses a salt or pH gradient, a factor that may alter MS sensitivity throughout the run, but has proven successful for biopharmaceutical applications . HIC has emerged as a promising alternative for analysis of intact proteins in low charge states and has most notably been applied to characterization of intact proteins and monoclonal antibodies at the MS1 level and/or in conjunction with ECD fragmentation. ,, The exact mechanism of low-charge-state production via HIC of intact proteins is not fully understood in the context of protein structures, and HIC has not yet been adapted for high-throughput nanoscale separations. One other emerging separation method for intact proteins is capillary electrophoresis, offering high separation efficiency and being well-adapted for native-like proteins owing to the use of a buffered solution for sample migration. Among these methods, HIC has a number of similarities to conventional reversed-phase methods, using hydrophobic stationary phases and also a salt to mediate interactions between the stationary phases and proteins.…”
Section: Introductionmentioning
confidence: 99%