Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE‐ESI‐MS/MS system

We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic‐labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope‐labeled lysine or arginine. The heavy‐labeled peptides were spiked in the HCP digests at known concentrations. After CZE‐ESI‐MS/MS analysis, the peaks of native and isotopic‐labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic‐labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R² values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 μg sample of digested HCPs. The target proteins were present at the 7–30 ppt level in the purified HCP samples.     

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry

Four methods were compared for analysis of host‐cell protein (HCP) impurities in a recombinant mAb. First, CZE‐MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE‐MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE‐ESI‐MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.     

Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed‐phase liquid chromatography (RPLC)–MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath‐flow interface for the analysis of complex proteome digests. Single‐shot CZE–MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state‐of‐the‐art nano ultrahigh‐pressure LC system.     

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.     

Application of capillary isotachophoresis-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

By employing a capillary ITP (CITP)/CZE-based proteomic technology, a total of 1795 distinct mouse Swiss-Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss-Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF-based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2-database reference set.     

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states. The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.     

Capillary-zone electrophoresis/fast-atom bombardment mass spectrometry: design of an on-line coaxial continuous-flow interface

An on-line coaxial continuous-flow capillary-zone electrophoresis/fast-atom bombardment mass spectrometry (CZE/FAB-MS) interface is described. This interface is shown to be capable of acquiring mass spectra in an on-line fashion from low femtomole amounts of peptides while maintaining high (hundreds of thousands of plates) electrophoretic separation efficiencies. Active electrophoretic transport of the analytes directly to the FAB probe tip obviates the need for a transfer line from the end of the CZE capillary to this point, and thereby precludes the zone broadening that would otherwise occur both within such a transfer line and in the connections between the CZE column and the transfer line. The capability of acquiring an on-line tandem mass spectrometry (MS/MS) spectrum of an electrophoretically separated analyte using this interface is also demonstrated.     

Development of a comprehensive multidimensional liquid chromatography system with tandem mass spectrometry detection for detailed characterization of recombinant proteins

A multidimensional comprehensive liquid-phase separation system (2DLC) coupled on-line to an electrospray-ionization time-of-flight (ESI-TOF) mass spectrometer (MS) was used to resolve structural alterations and/or post-translational modifications for detailed protein characterization. The system described in this work consists of cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. A unique spiked gradient was employed in the first dimension to enhance recovery of peptides. This combination of separation followed by MS detection offered the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. During the course of this study it was determined that altered or modified peptides were shown to be better resolved than during a one-dimensional separation. The 2DLC/ESI methodology allowed for a comprehensive evaluation of post-translational modifications and chemical reactions of recombinant proteins, providing a meaningful evaluation of product quality that was not possible with other current analytical approaches. In addition, the system can be used to provide sequence coverage of complex proteins.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Comprehensive two-dimensional chromatography and capillary electrophoresis coupled with tandem time-of-flight mass spectrometry for high-speed proteome analysis

A comprehensive two-dimensional capillary liquid chromatography and capillary zone electrophoresis system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) proteomics analyzer is presented. Protein/peptide samples were separated by capillary high-performance liquid chromatography (cHPLC). The effluents from cHPLC (the first dimension) were continuously transferred into capillary zone electrophoresis (CZE, the second dimension) through a novel valve-free hydrodynamic sampling interface. The CZE effluents were mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix sheath flow via CE-MALDI interface, and then directly deposited on the MALDI target at a 3 s time-interval for further MS analysis. The high efficiency of the overall system was demonstrated by analysis of proteins in D20 (human hepatocellular carcinoma model in nude mice with high metastatic potential) liver cancer tissue. More than 300 proteins were identified, which proved the system potential for high-throughput analysis and application in proteomics.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

MS2Assign,automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides

In a previous report (Young et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802-5806), we provided a proof-of-principle for fold recognition of proteins using a homobifunctional amine-specific chemical crosslinking reagent in combination with mass spectrometry analysis and homology modeling. In this current work, we propose a systematic nomenclature to describe the types of peptides that are generated after proteolysis of crosslinked proteins, their fragmentation by tandem mass spectrometry, and an automated algorithm for MS/MS spectral assignment called "MS2Assign." Several examples are provided from crosslinked peptides and proteins including HIV-integrase, cytochrome c, ribonuclease A, myoglobin, cytidine 5-monophosphate N-acetylneuraminic acid synthetase, and the peptide thymopentin. Tandem mass spectra were obtained from various crosslinked peptides using post source decay MALDI-TOF and collision induced dissociation on a quadrupole-TOF instrument, along with their automated interpretation using MS2Assign. A variety of possible outcomes are described and categorized according to the number of modified lysines and/or peptide chains involved, as well as the presence of singly modified (dead-end) lysine residues. In addition, the proteolysis and chromatographic conditions necessary for optimized crosslinked peptide recovery are presented.     

Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis

A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.     

Electrochemical properties of capillary electrophoresis-nanoelectrospray mass spectrometry

Electrochemical properties are inherent to the techniques of electrophoresis and electrospray ionization. Interfacing capillary zone electrophoresis (CZE) with electrospray mass spectrometry (ESMS) can lead to the observation of oxidized species generated as a result of the electrochemical nature of this coupling. Using a nanoelectrospray (nES) interface combined with CZE, controlled chemical oxidation of peptides is demonstrated. The electrolysis of water is used to explain the origin of the chemically oxidized species and this is confirmed using experiments with ¹⁸O labeled water. Identification of the oxidized residues was possible using tandem mass spectrometry to sequence the modified peptides. Methionine was found to be the most readily oxidized residue, followed by aromatic amino acids. Surprisingly, oxidation of aliphatic residues (leucine) was also observed. Addition of a reducing agent to the CZE buffer was found to reduce, but not eliminate, the extent of oxidation. The electrochemical generation of protons at the electrosprayer was used to assist in the analysis of monophosphate nucleotides. Nucleotides were separated as anions followed by detection as [M + H]⁺ ions.     

Heterogeneity of peptide adducts with carbonylated lipid peroxidation products

Highly reactive lipid peroxidation‐derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide–oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP–peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein–oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography–tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co‐incubated with oxidized 1‐palmitoyl‐2‐linoleoyl‐sn‐glycerophosphatidylcholine to characterize the collision‐induced dissociation behavior of peptide–carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4‐hydroxy‐2‐aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid‐bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision‐induced dissociation characteristics specific for each class of oxoLPP–peptide adducts. Copyright © 2015 John Wiley & Sons, Ltd.     

Capillary electrokinetic chromatography of insulin and related synthetic analogues

With the implementation of recombinant DNA technology in the pharmaceutical industry, some synthetic insulins have been developed in order to improve the therapy of diabetes. These analogues differ only slightly in the amino acid sequence, therefore displaying a great challenge for analytical chemistry. Within the work presented in this paper, capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) with sodium dodecylsulphate (SDS) as micelle-forming agent, and microemulsion electrokinetic chromatography (MEEKC) with microemulsions consisting of SDS, n-octane and 1-butanol were investigated for the separation of human insulin and five synthetic analogues. Best results were achieved with a solvent-modified MEKC system consisting of 100 mM sodium dodecyl sulphate and 15% acetonitrile in 10 mM borate buffer (pH 9.2). A similar system based on perfluorooctanoic acid as micelle-forming agent in ammonium acetate (pH 9.2) was successfully employed for the hyphenation with a quadrupole/time-of-flight mass spectrometer via a sheath-flow interface. In this case, detection limits at 10 mg/L could be achieved.     

Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.     

Selective enrichment and ultrasensitive identification of trace peptides in proteome analysis using transient capillary isotachophoresis/zone electrophoresis coupled with nano-ESI-MS

Besides the complexity in protein samples of biological origin, probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. As demonstrated in this work, transient capillary ITP/zone electrophoresis (CITP/CZE) provides selective analyte enrichment through electrokinetic stacking and extremely high resolving power toward protein and peptide mixtures. The result of the CITP process is that major components may be diluted, but trace compounds are concentrated. The on-column transition of CITP to CZE minimizes additional band broadening while providing superior analyte resolution. Online coupling of transient CITP/CZE with nano-ESI-MS allows ultrasensitive detection of trace peptides at levels of subnanomolar concentration or subfemtomole mass in complex peptide mixtures. More importantly, selective enrichment of trace peptides enables the identification and sequence analysis of low-abundance peptides co-migrated with highly abundant species at a concentration ratio of 1:500,000. The combined CITP/CZE-nano-ESI-MS system is demonstrated to be at least one to two orders of magnitude more sensitive than that attained in conventional electrophoretic and chromatographic-based proteome technologies over a wide dynamic concentration range, potentially allowing comprehensive analysis of protein profiles within a small cell population and limited tissue samples using conventional mass spectrometers. Furthermore, the speed of CITP/CZE separation and the lack of column equilibration in CITP/CZE not only improve the throughput of proteome analysis, but also facilitate its seamless integration with other separation technologies in a multidimensional protein identification platform.