Capillary Isoelectric Focusing Mass Spectrometry Research Articles

Deciphering nanoparticle protein coronas by capillary isoelectric focusing-mass spectrometry-based top-down proteomics.

The nanoparticle (NP) protein corona significantly influences the outcome of nanomedicine. We present the first example of top-down proteomics (TDP) measurement of the protein corona using capillary isoelectric focusing-mass spectrometry, identifying seventy proteoforms of 16 cancer-related genes. This technique has the potential to revolutionize our understanding of the protein corona and advance nanomedicine.

In-Depth Structure and Function Characterization of Heterogeneous Interchain Cysteine-Conjugated Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), integrating high specificity of antigen-targeting antibodies and high potency of cell-killing chemical drugs, have become one of the most rapidly expanding therapeutic biologics in oncology. Although ADCs were widely studied from multiple aspects, overall structural elucidation with comprehensive understanding of variants is scarcely reported. Here, for the first time, we present a holistic and in-depth characterization of an interchain cysteine-conjugated ADC, focusing on conjugation and charge heterogeneity, and in vitro biological activities. Conjugation mapping utilized a bottom-up approach, unraveled positional isomer composition, provided insights into the conjugation process, and elucidated how conjugation affects the physicochemical and biological properties of an ADC. Charge profiling combined bottom-up and top-down approaches to interrogate the origin of charge heterogeneity, its impact on function, and best practice for characterization. Specifically, we pioneered the utilization of capillary isoelectric focusing-mass spectrometry to decode not only critical post-translational modifications but also drug load and positional isomer distribution. The study design provides general guidance for in-depth characterization of ADCs, and the analytical findings in turn benefit the discovery and development of future ADCs.

Imaged capillary isoelectric focusing employing fluorocarbon and methylcellulose coated fused silica capillary for characterization of charge heterogeneity of protein biopharmaceuticals

AbstractIdentifying and characterizing charge heterogeneity is essential for the successful development and production of biopharmaceuticals. Imaged capillary isoelectric focusing technology based on isoelectric point differentiation has been becoming the gold standard of quality and manufactory process control in the biopharmaceutical industry due to its high‐resolution characterization of protein charge variants with high throughput. Fluorocarbon‐coated capillaries are widely used in the imaged capillary isoelectric focusing to suppress the electroosmotic flow and minimize the protein adsorption onto the fused silica capillary inner surface. Additionally, polymers such as methylcellulose are usually added to the sample solution to serve as a dynamic capillary coating that improves the peak shape and resolution during the imaged capillary isoelectric focusing separation, especially for complex proteins. However, the addition of methylcellulose tends to result in tedious operation, “spike peaks” from bubble generation, and frequent capillary blockage during the imaged capillary isoelectric focusing separation. In addition, methylcellulose is not compatible with mass spectrometry and easily produces the contamination of mass spectrometry ion source when carrying out imaged capillary isoelectric focusing—mass spectrometry direct coupling. Recently, a new imaged capillary isoelectric focusing method was developed employing a methylcellulose‐coated capillary cartridge, to avoid the addition of methylcellulose which is then present throughout the whole analysis. When applied to protein drug characterization the established imaged capillary isoelectric focusing method demonstrated high repeatability, stable coatings, outstanding separation efficiency, and excellent isoelectric point differentiation. In addition, we compared imaged capillary isoelectric focusing separation using the methylcellulose‐coated capillary with that utilizing the routinely coated capillary such as fluorocarbon, illustrating that methylcellulose coating provided consistent results and could be seamlessly integrated into an existing drug discovery process. Finally, the methylcellulose‐coated fused silica capillary was applied to imaged capillary isoelectric focusing—mass spectrometry for characterizing protein charge variants allowing reliable identification of mass spectrometry after imaged capillary isoelectric focusing separation. This can greatly simplify the operation steps and prevent the contamination of mass spectrometry ion source that often results from using routinely coated capillaries ultimately making this an essential innovation of imaged capillary isoelectric focusing—mass spectrometry that greatly improves the imaged capillary isoelectric focusing compatibility with mass spectrometry.

Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology.

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase-zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody-drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes.

An improved capillary isoelectric focusing-mass spectrometry method for high-resolution characterization of monoclonal antibody charge variants.

Routine and high-resolution characterization of monoclonal antibody (mAb) charge variants is vital for controlling mAb quality as therapeutics. Capillary isoelectric focusing-mass spectrometry (cIEF-MS) has emerged as a powerful tool for characterizing mAb charge variants because it can achieve high-resolution separation and highly sensitive detection of proteins. It provides much better identification of charge variants than the traditionally used cIEF-UV method. However, further improvement of cIEF-MS regarding stability and separation resolution is needed. Here, we improved the stability and enhanced separation resolution of automated cIEF-MS by bettering the quality of capillary neutral coating, reducing catholyte pH to 10 for cIEF-MS for the first time, and systematically optimizing the cIEF separation conditions. The improved cIEF-MS method was applied to characterize charge variants of three previously well characterized mAbs (NISTmAb, cetuximab, trastuzumab) and one tool mAb (mAb1). The charge variants of the studied mAbs were well resolved, and the majority of post-translational modifications (PTMs) found in those mAbs agreed with the literature. cIEF-MS analyses of mAb1 were capable of discovering ten charge variants with various interesting PTMs, such as PGK amidation, incomplete C-terminal lysine clipping, glycosylation, and deamination. cIEF-MS was successfully used for accurately determining the isoelectric points (pIs) of mAb1 charge variants via analyzing the pI markers and spiking in a standard protein (cytochrome c) to samples for migration time normalization, which is beneficial for evaluating pI-related pharmacokinetic properties. Our cIEF-MS agreed with and, in some cases (i.e., cetuximab and mAb1), outperformed cIEF-UV for detecting mAb charge variants.

Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry

Membranous nephropathy (MN) is one of the most common causes of primary glomerular diseases worldwide. The M-type phospholipase A2 receptor (PLA2R), an antigen expressed in more than 70% of cases of idiopathic membranous nephropathy (IMN), is a biomarker which is now used by physicians for clinical diagnosis. Despite the prevalence of PLA2R in the cases of MN, it is not always effective to use PLA2R for differentiating primary or secondary MNs. On the other hand, urinary albumin assay is one of the de facto tests for kidney function testing for several decades. In this work, urinary albumin species between primary and secondary MN patients are compared using a newly developed capillary isoelectric focusing – mass spectrometry (CIEF-MS) technology. The distinct patterns of cationic and acidic urinary albumin species, as revealed by this novel CIEF-MS technology, suggest potential applications of this differential analysis for subtyping of membranous nephropathy. Further investigation of these cationic human albumin species in urine may provide clues to the disease onset and development of MN, thus facilitating treatment. In addition, this novel workflow of using CIEF-MS for urinary protein analysis may be beneficial to the research, pathology, prognosis, and diagnosis of many other types of kidney diseases, such as chronic kidney disease, diabetic nephrology, etc.

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface.

Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF-MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF-MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF-MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights ( Mw) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact Mw values showed ppm level accuracy compared to theoretical predictions.

Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants.

We report a new online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for monoclonal antibody (mAb) charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source and a time-of-flight MS with pressure-assisted chemical mobilization. To develop a successful, reliable CIEF-MS method for mAb, we have selected and optimized many critical, interrelating reagents and parameters that include (1) MS-friendly anolyte and catholyte; (2) a glycerol enhanced sample mixture that reduced non-CIEF electrophoretic mobility and band broadening; (3) ampholyte selected for balancing resolution and MS sensitivity; (4) sheath liquid composition optimized for efficient focusing, mobilization, and electrospray ionization; (5) judiciously selected CIEF running parameters including injection amount, field strength, and applied pressure. The fundamental premise of CIEF was well maintained as verified by the linear correlation (R2 = 0.99) between pI values and migration time using a mixture of pI markers. In addition, the charge variant profiles of trastuzumab, bevacizumab, infliximab, and cetuximab, obtained using this CIEF-MS method, were corroborated by imaged CIEF-UV (iCIEF-UV) analyses. The relative standard deviations (RSD) of absolute migration time of pI markers were all less than 5% (n = 4). Triplicate analyses of bevacizumab showed RSD less than 1% for relative migration time to an internal standard and RSD of 7% for absolute MS peak area. Moreover, the antibody charge variants were characterized using the online intact MS data. To the best of our knowledge, this is the first time that direct online MS detection and characterization were achieved for mAb charge variants resolved by CIEF as indicated by a well-established linear pH gradient and correlated CIEF-UV charge variant profiles.

Simplified capillary isoelectric focusing with chemical mobilization for intact protein analysis.

We report a capillary isoelectric focusing system based on a sequential injection method for simplified chemical mobilization. This system was coupled to an ion trap mass spectrometer with an electrokinetically pumped nanoelectrospray interface. The nanoelectrospray emitter employed an acidic sheath electrolyte. To simplify focusing and mobilization, a plug of ammonium hydroxide was first injected into the capillary, followed by a section of mixed sample and ampholyte. During focusing, the NH3 H2 O section worked as catholyte. As focusing progressed, the NH3 H2 O section was titrated to lower pH by the acidic sheath electrolyte. Chemical mobilization started automatically once the ammonium hydroxide was consumed by the acidic sheath flow electrolyte, which then acted as the mobilization solution. In this report, the lengths of the NH3 H2 O section and sample were optimized. With a 1 m long capillary, a relative short plug of the NH3 H2 O section (3 cm) produced both fast migration and reasonable separation resolution. The simplified capillary isoelectric focusing mass spectrometry system produced base peak intensity relative standard deviation of 8.5% and migration time relative standard deviation ≤0.6% for myoglobin and cytochrome C in triplicate runs.

Optimization of protein identification from digests as analyzed by capillary isoelectric focusing-mass spectrometry

Capillary isoelectric focusing (CIEF) is a high-resolution separation technique for the analysis of peptides and protein digests. When coupled to ion trap-mass spectrometry (CIEF-MS) the unique separation mechanism is combined with a highly efficient detection system. In an earlier report, we described aspects of separation and interfacing in connection to the analysis of a digest of set of standard proteins. Now, we report on different aspects of the process of protein identification. Sequest software parameters were optimized by using a standard protein digest. These settings were used for the analysis of periplasmic proteins from Escherichia coli. Since in CIEF peptides are focused according to their pI values, the mobilization time of a particular peptide is dependent on its pI value. Based on this relation, the identification of some peptides was facilitated. Furthermore, the Sequest settings that were used could be evaluated. In total, 159 proteins were identified in a single run.

On-line capillary isoelectric focusing–mass spectrometry for quantitative analysis of peptides and proteins

On-line capillary isoelectric focusing–mass spectrometry (cIEF–MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF–MS employing 1% Pharmalyte 3–10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 μM each, Δp I = 0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1–5 μM) well correlated ( R = 0.999) with that obtained by the conventional RP–HPLC method. The limit of detection was 0.22 μM, which was about 10 times lower than that by UV detection (2 μM). The repeatability and accuracy were <15 and <11%, respectively. cIEF–MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity ( R 2 = 0.998) was also observed between the transferrin concentration (0.5–1.2 g/L) and peak area ratio (IS; β-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability (∼10% at 1 g/L).

Capillary isoelectric focusing‐mass spectrometry for shotgun approach in proteomics

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.

Capillary isoelectric focusing--mass spectrometry of proteins and protein complexes.

Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole-cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of noncovalent complexes on the intact complex level.

Capillary isoelectric focusing–mass spectrometry: analysis of protein mixtures from human body fluids

Isoelectric focusing within a fused silica capillary (cIEF) has proved to be a powerful and practical method for high-resolution separation of analytes from complex biological mixtures. This technique overcomes many of the problems of isoelectric focusing within slab gel media. However current cIEF systems commonly utilize UV detection which limits the detail of analyte structural information that is obtained during analysis. The use of mass spectrometry (MS) as the detection system provides much greater structural information about the detected analytes allowing accurate relative molecular mass (M(r)) determination for proteins and polypeptides. We have constructed a cIEF-MS interface and compared the separation of standard proteins analyzed by cIEF-UV with cIEF-MS. This allowed rapid optimization of the cIEF-MS system performance. Further we have demonstrated the use of MS as a detection system provides accurate M(r) information and can provide analyte modification details. These factors increase the likelihood of absolute identification for physiological proteins within complex in vivo-derived mixtures. To demonstrate the value of cIEF-MS in such analyses we have undertaken an examination of cerebrospinal fluid (CSF), and tentatively identified a number of constituent proteins. We have also analyzed whole blood from control and diabetic patients. We show that glycated alpha- and beta- chains of hemoglobin are found in almost equal abundance in diabetic patient blood. From these results we suggest cIEF-MS is an efficient and useful tool for the separation and examination of in vivo-derived analytes within physiological fluids.

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry.

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.

On-line desalting of physiologically derived fluids in conjunction with capillary isoelectric focusing-mass spectrometry

Capillary isoelectric focusing (cIEF) coupled to mass spectrometry (cIEF-MS) offers a potentially very powerful analytical tool for the investigation of physiological samples. The high resolving capabilities of cIEF in combination with the high sensitivity and enhanced structural information provided by MS is highly desirable for the analysis of complex samples. However a major limitation of the technique has always been the requirement to desalt samples prior to cIEF analysis. Such desalting normally occurs off-line and therefore adds complexity and the possibility of sample loss or contamination. In this study we demonstrate the use of a modified cIEF protocol which enables samples containing physiological levels of salts to be desalted on-line, within the cIEF capillary. This new technique is very fast and efficient, allowing the direct analysis of a physiologically derived fluid that contains a complex mix of proteins, such as human cerebrospinal fluid by cIEF-MS in a single step experiment.

A CE/ESI-MS interface for stable, low-flow operation.

A rugged, stable liquid sheath interfacing procedure for on-line CE/MS is described. This procedure combines optimized component sizes, tapered capillary tips, and adjustment of the capillary with respect to the liquid sheath tube during electrospray operation (active capillary positioning) to establish a stable electrospray quickly and reliably. The interface is especially effective at low flow rates, and CE/MS with a liquid sheath flow rate of 250 nL/min has been achieved. Active capillary positioning also allows on-line capillary isoelectric focusing mass spectrometry with in-probe focusing. In this application, the capillary is retreated into the liquid sheath tube, which becomes a microreservoir. After focusing, the capillary is returned to its proper position for electrospray, and focused zones are mobilized into the mass spectrometer for on-line detection. Both active capillary positioning and in-probe focusing readily lend themselves to automation.