Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high‐resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ≥100 fold excess of fluorophore reagent, 8‐aminopyrene‐1,3,6‐trisulfonic‐acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:≥100 ratio) maintained the >95% derivatization yield at 55°C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8‐aminopyrene‐1,3,6‐trisulfonic‐acid simplified the removal of excess reagent, important in both CE‐LIF (electrokinetic injection bias) and CE‐MS (ion suppression). Coupling CE‐ ESI‐MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

Novel monofunctional and bifunctional boronic acid‐functionalized squarylium dyes as precolumn and on‐column labels for protein analysis by capillary electrophoresis with laser‐induced fluorescence

This article presents a continuous capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) following spectral studies of the noncovalent interactions between novel Squarylium Boronic Acid 4 (SQ‐BA4) & Squarylium Diboronic Acid 2 (SQ‐DBA2) squarylium dyes and human serum albumin (HSA). Two protocols were used wherein the on‐column‐labeling protocol was found to be more sensitive than the precolumn one by showing a better enhancement in the peak area of the HSA–dye complex besides lower limits of detection (LODs) for HSA. Also, stability studies were conducted with or without HSA using precolumn‐labeling mode over one week exhibiting the superiority of SQ‐BA4 to SQ‐DBA2. Then, a mixture containing three model proteins, HSA, β‐lactoglobulin B, and transferrin, was labeled on‐column with both dyes and completely resolved by CE‐LIF after optimization of several parameters. Both dyes provided lower LODs for HSA than those of β‐lactoglobulin B and transferrin with higher sensitivities. In addition, the SQ‐BA4 dye showed again greater sensitivities with all the three proteins than SQ‐DBA2.     

Cover Picture: Electrophoresis 2'2011

Issue no. 2 is a regular issue assembled of 16 solid and original research articles distributed over 3 distinct parts. Part I is on novel trends in fundamentals and methodologies including theoretical models for selectivity of charged solutes in MEKC, system peaks in indirect detection, measuring epimerization constants by MEEKC, bundled CE using micro‐structured fibers, 2‐D separations by coupling CIEF and CEC, high speed DNA CE, MCE of N‐glycans and mucin expression in a microfluidic gradient device. Part II is concerned with detection, sensitivity enhancement, on‐column preconcentration and microdialysis sampling involving the design of continuous full filling CEC‐ESI‐MS using nanoparticles, CE‐fluorescence using tapered optical fiber, CZE separation of pesticide residues in water samples with acid‐assisted on‐column preconcentration and CE‐LIF to detect neurotransmitter amino acids and carbamathione in brain microdialysis samples. Novel methods for the separation and profiling of various proteins and large nucleic fragments are described in 4 consecutive papers grouped in part III. Featured articles include: Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography (( 10.1002/elps.201000405 )) Bundled capillary electrophoresis using microstructured fibres ( 10.1002/elps.201000442 )) Two‐dimensional separation system by on‐line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping ( 10.1002/elps.201000419 )) Microchip electrophoresis of N‐glycans on serpentine separation channels with asymmetrically tapered turns ( 10.1002/elps.201000461 ))     

A simple microfluidic system for efficient capillary electrophoretic separation and sensitive fluorimetric detection of DNA fragments using light-emitting diode and liquid-core waveguide techniques

A miniaturized CE system has been developed for fast DNA separations with sensitive fluorimetric detection using a rectangle type light-emitting diode (LED). High sensitivity was achieved by combining liquid-core waveguide (LCW) and lock-in amplification techniques. A Teflon AF-coated silica capillary on a compact 6x3 cm baseplate served as both the separation channel for CE separation and as an LCW for light transmission of fluorescence emission to the detector. An electronically modulated LED illuminated transversely through a 0.2 mm aperture, the detection point on the LCW capillary without focusing, and fluorescence light was transmitted to the capillary outlet. To simplify the optics and enhance collection of light from the capillary outlet, an outlet reservoir was designed, with a light transmission window, positioned directly in front of a photomultiplier tube (PMT), separated only by a high pass filter. Automated sample introduction was achieved using a sequential injection system through a split-flow interface that allowed effective release of gas bubbles. In the separation of a phiX174 HaeIII DNA digest sample, using ethidium bromide as labeling dye, all 11 fragments of the sample were effectively resolved in 400 s, with an S/N ratio comparable to that of a CE system with more sophisticated LIF.     

Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis

DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive 32P-postlabeling method, which allows the detection of one adduct in 10(10) unmodified nucleotides in microg amounts of DNA. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation procedures. In addition, the simultaneous detection of adducts from different classes of carcinogens in a DNA sample is difficult. In order to overcome these drawbacks, we are developing a new detection method, comprising fluorescence labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with fluorescently labeled standard compounds such as unmodified nucleotides or alkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dyes based on the phosphoramidite technology for DNA synthesis. The separation of N3-methylated, N3-, O2- and O4-butylated thymidines from the unmodified nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.     

Universal proteolysis and MSn for N‐ and O‐ glycan branching analysis

The continually growing list of critical glycosylation‐related processes has made analytical methodology for detailed glycan characterization an area of increasing interest. Glycosylation is a post translational modification of unsurpassed complexity due to the variety of compositions and linkages formed by these biopolymers. Structural characterization of glycan isomers has been achieved using ion trap mass spectrometry and MSⁿ of released, permethylated glycans. However, N‐ and O‐glycans require different sample preparation strategies; and release of the glycans may be hindered, result in degradation of the glycan, and/or produce limited yields of permethylated product. In the current report, we demonstrate universal proteolysis of both N‐ and O‐linked glycoproteins to individual glycoamino acids. These samples were shown to be directly amenable to permethylation and MSⁿ analysis for isomeric structural determination. Universal proteolysis and permethylation provides an identical sample preparation strategy for both classes of glycans that avoids potential pitfalls of commonly used release methods. This methodology should be applicable to all glycoproteins and serve as an alternative to glycan release for MSⁿ branching analysis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Metabolic Labeling and Imaging of N‐Linked Glycans in Arabidopsis Thaliana

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N‐azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N‐acetylglucosamine (GlcNAc) and incorporated into N‐linked glycans, and possibly intracellular O‐GlcNAc. Click‐labeling with fluorescent probes enables visualization of newly synthesized N‐linked glycans. N‐glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N‐glycosylation is regulated in a developmental stage‐dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N‐linked glycosylation in plants.     

On‐line coupled capillary isotachophoresis‐capillary zone electrophoresis in hydrodynamically closed separation system hyphenated with laser induced fluorescence detection

An analytical method, based on a column coupling capillary ITP and CZE in a hydrodynamically closed separation mode hyphenated with the detection in the modular arrangement, was developed in this work. Analytical possibilities of this approach are demonstrated on the direct and ultrasensitive quantitative determination of quinine (QUI) in diluted real multicomponent ionic matrices (beverages, urine). The detection cell interface, with the rectangular arrangement of the optical channels inside, connected the separation capillary with the LIF detector via optical fibers in the on‐column detection arrangement. ITP enabled the direct large volume (30 μL) injections of the diluted real matrices with an on‐line sample pretreatment (preseparation, preconcentration) so that no external sample preparation (except for the dilution) was necessary for the separation of the analyte in the multicomponent ionic matrices. Due to the ITP sample preconcentration and intrinsic sensitivity of the LIF detection, very low concentration LOD (as low as 77 pg/mL), were reached at the same time. This was ca. two orders lower than the corresponding LOD achieved by the same 2D separation system with UV absorbance detection. Compared to the single column CE‐LIF methods applied for this model analyte and matrix, this method was found to be superior in terms of concentration LOD, with acceptable selectivity and benefits of the on‐line sample preparation. A food control and bioanalytical application clearly illustrates great practical possibilities and routine use of the proposed modular ITP–CZE–LIF technique.     

Poly(amidoamine) dendrimer‐coated magnetic nanoparticles for the fast purification and selective enrichment of glycopeptides and glycans

Glycosylated proteins modulate various important functions of organisms. To reveal the functions of glycoproteins, in‐depth characterization studies are necessary. Although mass spectrometry is a very efficient tool for glycoproteomic and glycomic studies, efficient sample preparation methods are required prior to analyses. In the study, poly(amidoamine) dendrimer‐coated magnetic nanoparticles were presented for the specific enrichment and fast purification of glycopeptides and glycans. The enrichment and purification performance of the developed method was evaluated both at the glycopeptide, and the glycan level using several standard glycoprotein digests and released glycan samples. The poly(amidoamine) dendrimer‐coated magnetic nanoparticles not only showed selective affinity (Immunoglobulin G/Bovine Serum Albumin, 1/10 by weight) to glycopeptides and released glycans but also good sensitivity (0.4 ng/µL for Immunoglobulin G) for glycoproteomic and glycomic applications. Thirty‐five glycopeptides of Immunoglobulin G were detected after enrichment with poly(amidoamine) dendrimer‐coated magnetic nanoparticles. In addition, 55 ¹⁸O tagged deamidated glycopeptides belonging to human plasma glycoproteome were confirmed. Finally, fifty 2‐aminobenzoic acid, and 30 procainamide‐labelled human plasma N‐glycans released from human plasma glycoproteins were determined after purifications. The results indicate that the proposed enrichment and purification method using poly(amidoamine) dendrimer‐coated magnetic nanoparticles could be simply adjusted to sample preparation methods.     

Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.     

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid-assisted capillary electrophoresis

CE separations of glycans taken from the cancer drug, Trastuzumab (Herceptin(?)), were accomplished using phospholipid additives. Glycans were labeled with 1-aminopyrene-3,6,8-trisulfonic acid and were separated with efficiencies as high as 510000 theoretical plates in a 60.2 cm 25 μm id fused-silica capillary. The thermally tunable phospholipid was loaded into the capillary when it possessed a viscosity similar to that of water. The temperature was increased, and the separations were performed when the material exhibited higher viscosity. Enzymes were integrated into the separation with the phospholipid additive. Neuraminidase, β1-4 galactosidase, and β-N-acetylglucosaminidase were injected into the capillary without covalent modification and used for enzyme hydrolysis. Exoglycosidase enzymes cleaved the terminal glycan residues. The glycan sequence could be verified based on enzyme specificity. Neuraminidase was used to determine total glycan content of the low-abundance glycans containing sialic acid. β1-4 Galactosidase and β-N-acetylglucosaminidase were used sequentially in-capillary, to determine the structure of the high-abundance glycans.     

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.     

Laser‐Induced Fluorometry for Capillary Electrophoresis

Laser‐induced fluorometry (LIF) has achieved the detection of single molecules, which ranks it among the most sensitive of detection techniques, whereas capillary electrophoresis (CE) is known as a powerful separation method with resolution that is beyond the reach of many other types of chromatography. Therefore, a coupling of LIF with CE has established an unrivaled analytical technique in terms of sensitivity and resolution. CE‐LIF has demonstrated excellent performance in bioanalytical chemistry for the high‐resolution separation and highly sensitive detection of DNAs, proteins, and small bioactive molecules. This review describes the CE‐LIF methods developed by the author's group that include indirect and direct detection using diode lasers, post‐column derivatization, and Hadamard transformation, as well as applications to the binding assays of specific DNA immunoassays of proteins and to the determination of anticancer drugs.     

Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.     

Analysis of Bacillus globigii spores by CE

It is imperative in today's world that harmful airborne or solution-based microbes can be detected quickly and efficiently. Bacillus globigii (Bg) spores are used as a simulant for Bacillus anthracis (Ba) due to their similar shape, size, and cellular makeup. The utility of CE to separate and detect low levels of Bg spore concentrations will be evaluated. To differentiate spores from background particulates, several dyes, including fluorescamine, C-10, NN-127, Red-1c, and indocyanine green (ICG), were utilized as noncovalent labels for proteins on the Bg spore surface, as well as for HSA and homoserine standards. On-column labeling, with dye present in the running buffer, was utilized to obtain greater sensitivity and better separation. CE with LIF detection enables interactions between the dye and spore surface proteins to be observed, with enhanced fluorescence occurring upon binding of the dye to surface protein. Resulting electropherograms showed unique fingerprints for each dye with Bg spores. Migration times were under 10 min for all dye-spore complexes, with net mobilities ranging from 3.5x10(-4) to 6.9x10(-4) cm(2) V(-1) s(-1), and calibration curves yielded correlation coefficients of 0.98 or better for four of the dyes studied.     

Handling and detection of 0.8 amol of a near-infrared cyanine dye by capillary electrophoresis with laser-induced fluorescence detection

We are interested in the detection of DNA adducts and other trace analytes by labeling them with a fluorescent tag followed by use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for high resolution and sensitivity. Towards this goal, here we report the following: (1) synthesis and handling properties of a near-IR, carboxyl-substituted heptamethine cyanine dye; (2) modification of an existing ball lens LIF detector to provide near-LIF detection with excitation at 785 nm for CE; and (3) corresponding handling and detection of as little as 0.8 amol of the dye by enrich-injection of 4.7 microl of 1 x 10(-13) mol/l dye in methanol from an 8-microl volume into a corresponding CE-LIF system. The electrolyte for the separation was methanol-40 mmol/l aqueous sodium borate (98:2, v/v). This finding encourages further exploration of the dye by functionalization of its carboxyl group for chemical labeling purposes.     

Recent advances in affinity capillary electrophoresis

Use of the specificity of (bio)interactions can effectively overcome the selectivity limitation faced in capillary electrophoresis (CE), and the resulting technique usually is referred to as affinity capillary electrophoresis (ACE). Despite the high selectivity of ACE, several important problems still need to be addressed. A major issue in all CE separations, including ACE, is the concentration detection limit. Using UV detection, this is usually in the order of 10(-6) M whereas laser-induced fluorescence (LIF) detection can provide detection limits down to the sub-10(-10) M range. However, a marked disadvantage of LIF is that labeling of the analytes is usually required, which might change the interaction behavior of the solutes under investigation. Additionally, labeling reactions at sub-10(-10) M concentration levels are certainly not trivial and often difficult to perform quantitatively. Alternative and universal detection approaches, particularly mass spectrometric (MS) detection, look very promising but (A) CE-MS techniques are still far from routine application. Important future progress in sensitive detection strategies is likely to increase the use of ACE in the future.     

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.     

Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o‐phthalaldehyde

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high‐throughput analysis with flow‐gated CE. Here, we report a fast fluorescein‐labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow‐gated CE. This scheme was based on the reaction between primary amines and o‐phthalaldehyde in the presence of a fluorescent thiol, 2‐((5‐fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE‐SH). The short reaction time (<30 s) was suited for on‐line mixing and derivatization that was directly coupled with flow‐gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE‐SH, Tris(2‐carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow‐gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on‐chip labeling of proteins retained on support in SPE.