Integrated Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry System with an Immobilized Trypsin Microreactor for Online Digestion and Analysis of Picogram Amounts of RAW 264.7 Cell Lysate

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE–ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE–ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity

Integrated Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry System with an Immobilized Trypsin Microreactor for Online Digestion and Analysis of Picogram Amounts of RAW 264.7 Cell Lysate

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE–ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE–ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity

Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome <i>c</i>, myoglobin, bovine serum albumin (BSA), and β-casein) was used as the sample. The CZE–ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline-separated within 12 min. The limits of detection [signal-to-noise ratio (S/N) = 3] of the four model proteins ranged from 20 (cytochrome <i>c</i>) to 800 amol (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE–ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified

Bottom-Up Proteomics of <i>Escherichia coli</i> Using Dynamic pH Junction Preconcentration and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry

We report the use of the dynamic pH junction based capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) for bottom-up proteomics with an electrokinetically pumped sheath-flow nanospray capillary electrophoresis-mass spectrometry (CE-MS) interface and both LTQ-XL and LTQ-Orbitrap-Velos mass spectrometers. Conventional injection of 20 nL of a 1 mg/mL BSA digest identified 37 peptides and produced 66% sequence coverage. In contrast, pH junction injection of 130 nL (or larger) of a 0.05 mg/mL BSA digest identified 40 peptides and produced 70% coverage using a pH 6.5 sample buffer and the LTQ. A 20 nL conventional injection of a 1 mg/mL <i>Escherichia coli</i> digest identified 508 peptides and 199 proteins with the LTQ. A 400 nL pH junction injection of a 0.1 mg/mL <i>E. coli</i> digest identified 527 peptides and 179 proteins with the LTQ. Triplicate technical replicates of a 0.01 mg/mL sample with 400-nL injection volume using a pH junction identified 288 ± 9 peptides and 121 ± 5 proteins with the LTQ. There was outstanding concordance in migration time between the pH junction and normal injection. The pH junction produced narrower peaks and significant concentration for all but the most acidic components in the sample. Compared with the conventional stacking method, the pH junction method can generate comparable performance for small injection volume (20 nL) and significantly better concentration performance for a large injection volume (200 nL). We also applied the pH junction to three intact standard proteins and observed a >10× increase in peak intensity compared to conventional injection

Single-Shot Proteomics Using Capillary Zone Electrophoresis–Electrospray Ionization-Tandem Mass Spectrometry with Production of More than 1 250 <i>Escherichia coli</i> Peptide Identifications in a 50 min Separation

Capillary zone electrophoresis (CZE)–electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was optimized and applied for analysis of 1–100 ng <i>Escherichia coli</i> protein digests in a single run (single-shot analysis). The system employed an electrokinetically pumped nanospray interface, a coated capillary, and stacking conditions for sample injection. More than 1 250 peptides were identified by optimized single-shot CZE–ESI-MS/MS with 100 ng digest loaded and 50 min analysis time. When 10 ng and 1 ng digests were loaded, about 1 000 and 600 peptides were identified in a single-shot analysis, respectively. Compared with single-shot ultraperformance liquid chromatography (UPLC)–ESI-MS/MS, CZE–ESI-MS/MS produced fewer peptide IDs (1 377 ± 128 vs 1 875 ± 32) for large sample loading amounts (100 ng) with the same mass spectrometer time (50 min). However, when the loaded digest was mass limited (1 ng), CZE–ESI-MS/MS generated many more peptide identifications than UPLC–ESI-MS/MS (627 ± 38 vs 342 ± 113). In addition, CZE–ESI-MS/MS and UPLC–ESI -MS/MS provided complementary peptide level identifications. These results suggest that CZE–ESI-MS/MS may be useful for large-scale, comprehensive, and confident proteomics analysis

Single-Shot Proteomics Using Capillary Zone Electrophoresis–Electrospray Ionization-Tandem Mass Spectrometry with Production of More than 1 250 <i>Escherichia coli</i> Peptide Identifications in a 50 min Separation

Capillary zone electrophoresis (CZE)–electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was optimized and applied for analysis of 1–100 ng Escherichia coli protein digests in a single run (single-shot analysis). The system employed an electrokinetically pumped nanospray interface, a coated capillary, and stacking conditions for sample injection. More than 1 250 peptides were identified by optimized single-shot CZE–ESI-MS/MS with 100 ng digest loaded and 50 min analysis time. When 10 ng and 1 ng digests were loaded, about 1 000 and 600 peptides were identified in a single-shot analysis, respectively. Compared with single-shot ultraperformance liquid chromatography (UPLC)–ESI-MS/MS, CZE–ESI-MS/MS produced fewer peptide IDs (1 377 ± 128 vs 1 875 ± 32) for large sample loading amounts (100 ng) with the same mass spectrometer time (50 min). However, when the loaded digest was mass limited (1 ng), CZE–ESI-MS/MS generated many more peptide identifications than UPLC–ESI-MS/MS (627 ± 38 vs 342 ± 113). In addition, CZE–ESI-MS/MS and UPLC–ESI -MS/MS provided complementary peptide level identifications. These results suggest that CZE–ESI-MS/MS may be useful for large-scale, comprehensive, and confident proteomics analysis

Capillary Isoelectric Focusing-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Proteomic Analysis of Differentiating PC12 Cells By Eight-Plex Isobaric Tags for Relative and Absolute Quantification

We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry (ESI-MS/MS) approach identified 835 protein groups and produced 2 329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1 369 protein groups and 3 494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast

We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with a trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. From this, 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than that of previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified

Coupling Methanol Denaturation, Immobilized Trypsin Digestion, and Accurate Mass and Time Tagging for Liquid-Chromatography-Based Shotgun Proteomics of Low Nanogram Amounts of RAW 264.7 Cell Lysate

We report the shotgun proteomic analysis of mammalian cell lysates that contain low nanogram amounts of protein. Proteins were denatured using methanol, digested using immobilized trypsin, and analyzed by UPLC-ESI-MS/MS. The approach generated more peptides and higher sequence coverage for a mixture of three standard proteins than the use of free trypsin solution digestion of heat- or urea-denatured proteins. We prepared triplicate RAW 264.7 cell lysates that contained 6, 30, 120, and 300 ng of protein. An average of 2 ± 1, 23 ± 2, 134 ± 11, and 218 ± 26 proteins were detected for each sample size, respectively. The numbers of both protein and peptide IDs scaled linearly with the amount of sample taken for analysis. Our approach also outperformed traditional methods (free trypsin digestion of heat- or urea-denatured proteins) for 6–300 ng RAW 264.7 cell protein analysis in terms of number of peptides and proteins identified. The use of accurate mass and time (AMT) tags resulted in the identification of an additional 16 proteins based on 20 peptides from the 6 ng cell lysate prepared with our approach. When AMT analysis was performed for the 6 ng cell lysate prepared with traditional methods, no reasonable peptide signal could be obtained. In all cases, roughly ∼30% of the digested sample was taken for analysis, corresponding to the analysis of a 2 ng aliquot of homogenate from the 6 ng cell lysate

Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome <i>c</i>, myoglobin, bovine serum albumin (BSA), and β-casein) was used as the sample. The CZE–ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline-separated within 12 min. The limits of detection [signal-to-noise ratio (S/N) = 3] of the four model proteins ranged from 20 (cytochrome <i>c</i>) to 800 amol (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE–ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified