Electrospray ionization stability and concentration sensitivity in capillary electrophoresis-mass spectrometry using a flow-through microvial interface

Concentration sensitivity is a key performance indicator for analytical techniques including for capillary electrophoresis-mass spectrometry (CE–MS) with electrospray ionization (ESI). In this study, a flow-through microvial interface was used to couple CE with MS and improve the ESI stability and detection sensitivity. By infusing a peptide mixture through the interface into an MS detector at a typical flow rate for CE-MS analysis, the spatial region near the interface was mapped for MS signal intensity. When the sprayer tip was within a 6 × 6.5 × 5 mm region in front of the MS inlet, the ESI was stable with no significant loss of signal intensity for ions with m/z 239. Finite element simulations showed that the average electric field strength at the emitter tip did not change significantly with minor changes in emitter tip location. Experiments were conducted with four different mass spectrometer platforms coupled to CE via the flow-through microvial interface. Key performance indicators, that is, limit of detection (LOD) and linearity of calibration curves were measured for nine amino acids and five peptides. Inter- and intraday reproducibility were also tested. The results were shown to be suitable for quantification when internal standards were used.     

毛细管电泳质谱联用技术及其应用

本文介绍了用于毛细管电泳质谱联用仪器的多种接口技术，描述了ＣＺＥ，ＣＩＥＦ，ＣＧＥ，ＭＥＫＣ和ＣＩＴＰ等毛细管电泳技术和四极质谱，离子阱质谱，傅 叶变换离子回旋共振质谱，飞行时间质谱，磁质谱，解吸质谱等联用的现状及发展前景，对近年来ＣＥ－ＭＳ在酶解产物。蛋白质和肽，核苷酸，药物及代谢产物等领域中的应用作了详细述评。     

Design of a sheathless capillary eletrophoresis-mass spectrometry probe for operation with a Z-spray ionization source

The construction of a sheathless interface for capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS), for operation with a Z-Spray source on a Micromass Quattro-LC triple quadrupole mass spectrometer is described. Designing the interface involved machining a probe compatible with the setup already in place on the mass spectrometer, i.e., MegaFlow-Z ESI. The probe was made of Lexan with the same dimensions as the ESI probe supplied with the instrument. The electrical connection at the electrospray end of the CE capillary was made possible by gold-coating (sheathless CE-ESI-MS). The probe design as well as the electrical and power supply requirements are described in detail. Experiments were performed using this interface, and CE separations of mixtures containing pmole and sub-pmole amounts of peptides were monitored by on-line MS. For a standard peptide mixture (10(-4) M), separation efficiency was typically characterized by N > 10(4) theoretical plates with S/N > 400. Using the same experimental setup, it was also possible to conduct on-line CE-ESI-tandem MS (MS/MS) experiments on the same peptide mixture, and to determine the sequence of the peptides. MS/MS scan functions for different precursor ions were used either alternately or sequentially and the results from both methods were compared. The possibility of peptide mass mapping was explored, and CE-ESI-MS results were obtained for the digestion products of equine myoglobin. Separation efficiencies and S/N values were similar to those obtained for standard peptides. A complete map of the digestion products was obtained.     

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire that is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N = 3) was achieved for detection of myoglobin utilizing a 75-µm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550–1300 Da. The advantages of this new design include (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.     

Development of a sheathless CE‐ESI‐MS interface

A sheath‐flow interface is the most common ionization technique in CE‐ESI‐MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE‐MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath‐flow CE‐MS. Because the interface is easy to construct, it is cost‐effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE‐MS‐based metabolomic analysis.     

Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn‐superoxide dismutase

The native form of Cu,Zn‐superoxide dismutase (SOD‐1) is a homodimer that coordinates one Cu²⁺ and one Zn²⁺ per monomer. Cu²⁺ and Zn²⁺ ions play crucial roles in enzyme activity and structural stability, respectively. In addition, dimer formation is essential for SOD‐1 functionality, and in humans several SOD‐1 mutant isoforms have been associated with certain types of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder. In this paper we used capillary electrophoresis and mass spectrometry to study the different structures of bovine SOD‐1. The metal ions of the native enzyme (Cu₂,Zn₂‐dimer SOD‐1) were released in acidic medium in order to obtain apo‐SOD‐1, which is a monomer. Both substances were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and capillary electrophoresis with ultraviolet and electrospray ionization mass spectrometry detection (CE/UV and CE/ESI‐MS, respectively). With MALDI‐TOF‐MS, using matrices of sinapinic acid (SA) or 2,5‐dihydroxybenzoic acid (DHB) with or without trifluoroacetic acid (TFA), similar mass spectra were obtained for the metalated and non‐metalated samples. In both cases, an average molecular mass corresponding to the apo‐monomer SOD‐1 was calculated. This finding indicated that the metals were released from the Cu₂,Zn₂‐dimer SOD‐1 during sample preparation or ionization. For CE/UV and CE/ESI‐MS, two background electrolytes (BGEs) potentially compatible with ESI‐MS detection were used, namely 1 M of acetic acid (pH 2.3) and 10 mM of ammonium acetate (pH 7.3). Using a sheath liquid of 2‐propanol/water (60:40 v/v), with or without 0.1% v/v of formic acid, CE/ESI‐MS sensitivity was enhanced when the acidic BGE and the acidic sheath liquid were used. However, the electrophoretic profiles and the mass spectra obtained suggested that the metals of Cu₂,Zn₂‐dimer SOD‐1 were released, which generated the apo‐monomer during the electrophoretic separation. The neutral BGE provided enhanced conditions for the detection of the native enzyme. The differences between the mass spectra obtained for the Cu₂,Zn₂‐dimer and the apo‐monomer forms were significant and the presence of formic acid in the sheath liquid affected only sensitivity. Our results highlight the importance of selecting appropriate non‐denaturing separation and detection conditions to obtain reliable structural information about non‐covalent protein complexes by CE/ESI‐MS. Copyright © 2010 John Wiley & Sons, Ltd.     

Capillary electrophoresis-laser induced fluorescence-electrospray ionization-mass spectrometry: a case study

The simultaneous hyphenation of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and electrospray ionization-mass spectrometry (ESI-MS) as a novel combined detection system for CE is presented. beta-Carbolines were chosen as model analytes with a forensic background. Nonaqueous CE as well as conventional CE with an aqueous buffer system are compared concerning efficiency and obtainable detection limits. The distance between the optical detection window and the sprayer tip was minimized by placing the optical cell directly in front of the electrospray interface. Similar separation efficiencies for both detection modes could thus be obtained. No significant peak-broadening induced by the MS interface was observed. The high fluorescence quantum yield and the high proton affinity of the model analytes investigated resulted in limits of detection in the fg (nmol/L) range for both detection methods. The analysis of confiscated ayahuasca samples and ethanolic plant extracts revealed complementary selectivities for LIF and MS detection. Thus, it is possible to improve peak identification of the solutes investigated by the use of these two detection principles.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

用于蛋白质分析的毛细管等电聚焦-电喷雾质谱接口的改进

蛋白质组学对其分析技术提出了大规模、高通量的要求 [1] .传统的等电聚焦 ( p I) -分子量 ( MW)双向电泳技术 ( 2 D- Gel)尽管在蛋白质组学研究中占有重要地位 ,但其操作繁杂、工作周期长 .Pandey等[1] 将毛细管等电聚焦 ( CIEF)与电喷雾质谱 ( ESIMS)联用 ,使得 p I和 MW两维分离鉴定技术变得简单迅速 .但 CIEF- MS的接口操作需中断高压和将毛细管阴极端插入电喷雾管 ,故引起分析蛋白质的散焦和不重现 .本工作改进了 CIEF- MS接口 ,采用毛细管阴极端和电喷雾针一体化的电喷雾接口 ,无需中断高压 ,实现了 CIEF- MS的在线联…     

A hydrodynamic injection approach for capillary electrophoresis using rotor–stator valves

Sample injection is a critical step in a capillary electrophoresis (CE) analysis. Electrokinetic injection is the simplest approach and is often selected for implementation in portable CE instruments. However, in order to minimize the effect of sample matrix upon the results of a CE analysis, hydrodynamic injection is preferred. Although portable CE instruments with hydrodynamic injection have been reported, injection has always been performed at the grounded end of the capillary. This simplifies fluidic handling but limits coupling with electrochemical detectors and electrospray ionization–mass spectrometry (ESI–MS). We demonstrated previously that injection at the high-voltage (HV) end of the capillary could be performed using an HV-compatible rotary injection valve (fixed-volume injection). However, the mismatch between the bore sizes of the channels on the rotor–stator valve and the separation capillary caused peak tailing and undesired mixing, impairing analytical performance. In this work, we present an HV-compatible hydrodynamic injection approach that overcomes the issues associated with the fixed-volume injection approach reported previously. The performance of the CE instrument was demonstrated by analyzing a mixture of 13 amino acids by CE coupled to laser-induced fluorescence, which showed relative standard deviations for peak area and migration time below 5% and 1%, respectively, for triplicate analysis. Additionally, replicate measurements of a mixture of amino acids, peptides, nucleobases, and nucleosides by CE coupled to electrospray ionization–mass spectrometry (CE–ESI–MS) were performed to evaluate peak tailing, and results were similar to those obtained with a commercial CE–ESI–MS setup.     

A simple fabrication method for tapered capillary tip and its applications in high‐speed CE and ESI‐MS

Fabrication of capillaries with tapered tips is an important technique that is required in many analytical chemistry areas, such as ESI‐MS, CE, electrochemical analysis, and microinjection. This paper describes a simple and effective grinding‐based fabrication method for capillaries with tapered tips. A novel grinding mode utilizing the combination of rotation and precession of an elastic capillary was developed, which significantly improved the controllability to the grinding process as well as the capillary tip shape. The capillary was fabricated by fixing it in an electric drill installed perpendicularly, and grind the capillary tip rotated around its own axis as well as the drill axis on sandpapers. Compared with conventional fabrication techniques for capillary tips, the present method is easy to control the capillary tip shape in routine laboratories without the requirement of expensive equipments or poisonous reagent (e.g. hydrofluoric acid (HF) solution). Various capillaries with different tip diameters and tip taper angles could be fabricated using the present method with good controllability and reproducibility. These capillaries were applied in high‐speed CE and ESI‐MS analysis to demonstrate the feasibility and potential of this fabrication method.     

Development of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics

The applicability of a capillary zone electrophoresis–electrospray ionisation tandem mass spectrometric (CZE–ESI-MS–MS) method for the separation of nine fluoroquinolones was investigated. Method optimisation involved systematic trouble-shooting starting with the type and duration of capillary pre-washing and conditioning, the choice of both the CE run buffer, MS sheath liquid, CE run potential, ESI spray voltage, sheath gas flow-rate, MS capillary voltage and CE capillary and MS capillary temperatures. Another extremely important factor was found to be the degree to which the CE capillary protrudes into the ESI chamber as well as whether or not sheath gas and spray voltage are employed during the CE injection or not. The importance of the latter has, to our knowledge, not been addressed elsewhere. Nine fluoroquinolones have been separated and detected in a single run by this technique.     

Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling

The hyphenation of capillary electrophoresis (CE) with mass spectrometry (MS) is a powerful method to obtain high efficient, sensitive, and selective analyses. The successful coupling with electrospray ionization (ESI) source requires closed electric circuits for both the CE separation and the ESI processes. A wide range of interfaces has been proposed to satisfy this requirement. Among them, the new high sensitivity porous sprayer based on a porous tip achieves the electric connection by inserting the capillary outlet made of a porous material into an ESI needle filled with a conductive liquid and independently grounded. This device is compatible with the minute flow rates exhibited in CE and therefore makes possible the use of a nano-electrospray behavior. In this work, this interface was evaluated for hyphenating a CE with a single quadrupole MS instrument for low molecular weight analytes. Investigations aimed at highlighting the most influent parameters thanks to a design of experiments, reaching the best performance in terms of sensitivity and stability. MS signal intensities of various pharmaceutical compounds (e.g. amphetamines, β-blockers) emphasized high sensitivity and efficiency, while repeatability, expressed as relative standard deviation of corrected heights and areas, was suitable for quantitative purposes (<5%).     

On-line CE-MS using pressurized liquid junction nanoflow electrospray interface and surface-coated capillaries

A simple and cost-effective laboratory-made liquid junction interface was used for coupling of CE with MS. In this device the capillary column and the spray tip were positioned in the electrode vessel containing appropriate spray liquid. The electrospray potential was applied on the electrode inside the liquid junction. A stable electrospray was produced at nanoliter per minute flow rates generated in the emitter tip without using an external pump. This arrangement provided high durability of the spray tip and independent optimization of the CE separation (use of coated capillaries) and ESI conditions. CE-MS analysis of mixtures of drugs, peptides, tryptic digests of proteins and biological fluids was optimized with respect to the effects of the distance between the separation capillary and electrospray tip and pressure applied on the liquid junction. The sensitivity of the system, in terms of the LOD (base peak monitoring) was below 10 ng/mL for the beta-blocker drugs and below 200 ng/mL for peptide analysis.     

Sheathless capillary electrophoresis electrospray ionization‐mass spectrometry interface based on poly(dimethylsiloxane) membrane emitter and thin conducting liquid film

This study develops a sheathless CE‐MS interface using a robust PDMS membrane emitter and liquid‐film electric conduction. A 3D mold was constructed for casting the device by using a one‐step casting procedure. The interface consisted of a 125 μm‐thick triangular emitter with a 50 μm‐diameter microchannel, a conducting reservoir, and a 375 μm‐diameter channel for assembling the separation capillary. The separation capillary was inserted into the 375 μm channel and connected to the emitter through the conducting reservoir. The electric contact for the CE outlet was established through a conductive liquid film in the space between the capillary terminus and the 375 μm channel. The one‐step casting procedure and using a membrane emitter instead of a tapered emitter produced an easily fabricated and robust interface. A stable electrospray was obtained from 30 to 350 nL/min. Analyzing a five‐peptide mixture in low‐EOF (60 nL/min) and high‐EOF (210 nL/min) conditions demonstrated the utility of the interface.     

Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface

An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500–1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-µm inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ESI-MS analysis of a human blood sample.     

Interface for coupling nonaqueous wide-bore capillary electrophoresis with mass spectrometry

A liquid-junction-type interface where a thin spraying capillary is inserted inside the separation capillary was constructed for coupling nonaqueous wide-bore capillary electrophoresis (CE) to mass spectrometry (MS). The robust structure of the interface provided fairly easy capillary handling. The study was carried out with uncoated CE capillaries of 200 and 320 microm inner diameter (ID). 1-Propanol-acetonitrile (80:20 v/v) with acetate electrolyte provided a low conducting medium for CE and good spraying conditions for electrospray ionization (ESI) without sheath-flow and drying gas. Methamphetamine, alprenolol, and levorphanol served as model compounds. Approximate detection limits with the 200 microm ID capillary were 35-265 ng/mL.     

Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection.

On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-microns I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15-20 mM) of ammonium acetate or ammonium formate, and high organic modifier content (5-50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0-0.04 microliter/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous 'infusion' of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.     

Sensitivity enhancement in capillary electrophoresis-mass spectrometry of anionic metabolites using a triethylamine-containing background electrolyte and sheath liquid

Analyte responses in CE‐ESI‐MS using negative ionization are frequently relatively low, thereby limiting sensitivity in metabolomics applications. In order to enhance the ionization efficiency of anionic metabolites, BGEs and sheath liquids (SLs) of various compositions were evaluated. Pressure‐induced infusion and CE‐MS experiments showed that addition of triethylamine (TEA) to the BGE and SL enhanced analyte intensities. A BGE consisting of 25 mM TEA (pH 11.7) and an SL of water–methanol (1:1, v/v) containing 5 mM TEA was selected, providing separation and detection of ten representative test metabolites with good reproducibility (migration time RSDs<1%) and linearity (R²>0.99). This BGE yielded lower limits of detection (0.7–9.1 μM) for most test compounds when compared with common CE‐MS methods using a BGE and SL containing ammonium acetate (NH₄Ac) (25 and 5 mM, respectively). CE‐MS of human urine revealed an average amount of 231 molecular features in negative ionization mode when TEA was used in the BGE and SL, whereas 115 and 102 molecular features were found with an NH₄Ac‐containing BGE and SL, employing a bare fused‐silica (BFS) and Polybrene‐dextran sulfate‐Polybrene (PB‐DS‐PB)‐coated capillary, respectively. With the CE‐MS method using TEA, about 170 molecular features were observed that were not detected with the NH₄Ac‐based CE‐MS methods. For more than 82% of the molecular features that were detected with the TEA as well as the NH₄Ac‐containg BGEs (i.e. common features), the peak intensities were higher using TEA with gain factors up to 7. Overall, the results demonstrate that BGEs and SLs containing TEA are quite favorable for the analysis of anionic metabolites in CE‐MS.     

Capillary electrophoresis-based assessment of nanobody affinity and purity

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced “nanobody” EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50 mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0–12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (K_d) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the K_d of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE–MS using a BGE of 100 mM acetic acid (pH 2.8) in combination with a polybrene–dextran sulfate–polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE–MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.