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/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ∼10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein-and especially peptide-level identifications of the evaluated CE-MS-and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/ MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ∼60 min, CE-MS/MS identified ∼2000 ± 53 proteins on average from a single injection of ∼8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2-and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.

Coupling High-Field Asymmetric Ion Mobility Spectrometry with Capillary Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry Improves Protein Identifications in Bottom-Up Proteomic Analysis of Low Nanogram Samples

“…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).…”

“…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).…”

“…The capabilities of CE-MS for proteomic profiling at ultrasensitive levels have been previously demonstrated by our lab and others. 15−17 peptides has been highlighted as an advantage in CE-MS analysis 17,18 due to the shift in electrophoretic mobility imparted by chemical modifications that alter peptide charge. 19 FAIMS has been reported to improve the identification of phosphorylated peptides by enhancing the signal-to-noise ratio.…”

Microanalysis of Brain Angiotensin Peptides Using Ultrasensitive Capillary Electrophoresis Trapped Ion Mobility Mass Spectrometry

“…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.…”

Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States

“…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.…”