The use of multidimensional liquid-phase separations and mass spectrometry for the detailed characterization of posttranslational modifications in glycoproteins

The goal of characterization of the proteome, while challenging in itself, is further complicated by the microheterogeneity introduced by posttranslational modifications such as glycosylation. A combination of liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) offers the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. In the current work, the combination of liquid-phase separations and mass spectrometry is demonstrated through the on-line coupling of electrospray ionization mass spectrometry (ESI-MS) and off-line coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). LC/ESI-MS yields real-time results while maintaining the separation obtained from the LC analysis. CE/MALDI TOF-MS offers high-mass detection and extremely low detection limits. The unique separation selectivity of CE relative to reversed-phase HPLC separations of the members of a glycopeptide family was used to develop an integrated multidimensional analysis achieved by the off-line coupling of LC, CE, and MALDI TOF-MS. To demonstrate the applicability of these techniques to the characterization of the heterogeneity of posttranslational modifications present in glycoproteins, we will report on the study of the glycoforms present in a N-linked site in a single-chain plasminogen activator (DSPAα1).     

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.     

Characterization of the microdialysis junction interface for capillary electrophoresis/microelectrospray ionization mass spectrometry

A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESI-MS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency.     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

Isolation and identification of sialylated glycopeptides from bovine alpha1-acid glycoprotein by off-line capillary electrophoresis MALDI-TOF mass spectrometry

Sialylated glycopeptides contained in liquid chromatographic fractions of bovine alpha1-glycoprotein tryptic digests were isolated from asialo peptides using capillary electrophoresis (CE). CE effluents were deposited directly onto a metallic target and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This method allowed the characterization of four N-glycosylation sites in the glycoprotein, and each site was observed as a set of sialylated peptide glycoforms. Tandem mass spectrometry was used to confirm peptide sequences and glycan content in glycoforms. The CE method developed for this study resulted in a very clear separation of the sialylated from the asialo content of glycoprotein digests and proved very useful in the determination of the nature and location of sialylated glycans along the protein chain. This article is the first report describing the use of on-target CE fraction collection using a MALDI removable sample concentrator.     

Design and performance of a universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique

A split-flow capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) interface is introduced, in which the electrical connection to the CE capillary outlet is achieved by diverting part of the CE buffer out of the capillary through an opening near the capillary outlet. The CE buffer exiting the opening contacts a sheath metal tube which acts as the CE outlet/ESI shared electrode. In cases in which the ESI source uses a metal needle, the voltage contact to the CE buffer is achieved by simply inserting the outlet of the CE capillary, which contains an opening, into the existing ESI needle (thereby greatly simplifying the CE to MS interfacing). As a result of the concentration-sensitive nature of ESI, splitting a small percentage of the CE flow has minimal effect on the sensitivity of detection. In addition, because the liquid is flowing through the opening and out of the capillary, there is no dead volume associated with this interface. Moreover, bubble formation due to redox reactions of water at the electrode does not effect CE/ESI-MS performance, because the actual metal/liquid contact occurs outside of the CE capillary. The sensitivity associated with a sheathless CE/MS interface, the ease of fabrication, universality, and lack of any dead volume make this design a superior CE/ESI-MS interface. The performance of this interface is demonstrated by analyses of a peptide standard and a protein digest using a variety of capillary dimensions.     

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.     

Mass spectrometric characterization of functional poly(methyl methacrylate) in combination with critical liquid chromatography

State-of-the-art techniques for the mass spectrometric characterization of synthetic polymers have been applied to functional poly(methyl methacrylate), synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization. The polymers were first separated effectively according to functionality by liquid chromatography (LC) at the critical conditions (i.e., almost no influence of molecular weight on retention). The separated polymers were characterized off-line by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and both off-line and on-line by LC-electrospray-ionization-quadrupole-TOF-MS (LC-ESI-QTOF- MS). The on-line ESI experiments confirmed a clear baseline separation of the hydroxyl-functional prepolymers according to the number of hydroxyl groups. Labile end groups of PMMA, such as the dithioester group, were lost in the MALDI-TOF-MS experiments, while they were observed intact in the ESI-QTOF-MS spectra. This indicates that in the present case ESI is a much softer ionization technique than is MALDI. The ESI-MS experiments provided direct evidence that the RAFT polymers still exhibited living characteristics in the form of the dithio moiety.     

Development of a poly(dimethylsiloxane) interface for on-line capillary column liquid chromatography-capillary electrophoresis coupled to sheathless electrospray ionization time-of-flight mass spectrometry

An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 microL LC injection of 25 microg/mL peptide standard.     

Analysis of inorganic species by capillary electrophoresis-mass spectrometry and ion exchange chromatography-mass spectrometry using an ion spray source

Mixtures of inorganic ions separated by capillary electrophoresis (CE) and ion exchange chromatography (IC) are detected by mass spectrometry (MS) using an ion spray atmospheric pressure ionization source. The selectable degree of ion-adduct declustering and molecular fragmentation in the MS interface region allows the system to be operated as an elemental analyzer or as a molecular detector suitable for oxidation state determinations. Both inorganic anions and cations (including alkalis, alkaline earths, transition metals, and lanthanides) are analyzed by CE-MS. A variety of CE separation buffers are evaluated for the cation analyses (e.g., creatinine, ammonium acetate, and tris[hydroxymethyl]aminomethane). Only one of the buffers (i.e., creatinine) can be used for CE-indirect UV detection. A CE capillary permanently coated with strong anion exchange sites and a pyromellitic acid buffer (suitable for indirect UV detection) is used for the inorganic anion separations. The coated column eliminates the need for buffer modifiers to reverse the flow in the capillary, which then reduces background noise and mass spectral complexity. The separation and detection of 13 inorganic anions are also accomplished by IC using an anion exchange column with a carbonate-bicarbonate mobile phase, on-line suppressed conductivity detection, and mass spectrometric detection.     

CE/electrospray ionization-MS analysis of underivatized d/l-amino acids and several small neurotransmitters at attomole levels through the use of 18-crown-6-tetracarboxylic acid as a complexation reagent/background electrolyte

A new capillary electrophoresis/mass spectrometry technique is introduced for attomole detection of primary amines (including several neurotransmitters), amino acids, and their d/l enantiomers in one run through the use of a complexation reagent while using only approximately 1 nL of sample. The technique uses underivatized amino acids in conjunction with an underivatized capillary, which significantly reduces cost and analysis time. It was found that when (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TCA, MW 440) was used as the background electrolyte/complexation reagent during the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of underivatized amino acids, stable complexes were formed between the amino acids and the 18-C-6-TCA molecules. These complexes, which exhibited high ionization efficiencies, were detectable at attomole levels for most amino acids. The detection limits of the AA/18-C-6-TCA complexes were on the average more than 2 orders of magnitude lower than that of the free amino acids in solution. In addition to lower detection limits under CE/ESI-MS, a solution of 18-C-6-TCA in the concentration range of 5-30 mM provided high separation efficiency for mixtures of l-amino acids as well as mixtures of d/l-amino acids. By using a solution of 18-C-6-TCA as the background electrolyte in conjunction with an underivatized, 130-cm-long, 20-microm-i.d., 150-microm-o.d. fused-silica capillary and by monitoring the m/z range of the amino acid/18-C-6-TCA complexes (m/z 515-700), most of the standard amino acids and many of their enantiomers were separated and detected with high separation efficiency and high sensitivity (nanomolar concentration detection limits) in one run. The solutions of 18-C-6-TCA also worked well as the CE/ESI-MS BGE for low-level detection of several neurotransmitters and some of their d/l enantiomers as well as for the analysis of amino acids at endogenous levels in lysed red blood cells.     

Micellar electrokinetic chromatography-mass spectrometry using a polymerized chiral surfactant.

The coupling of chiral micellar electrokinetic chromatography (CMEKC) to mass spectrometry (MS) using conventional surfactant [above the critical micelle concentration (cmc)] is very challenging. Preliminary investigation in this laboratory indicates that the use of a chiral polymeric surfactant provides one possible solution to this difficult coupling. This is because of many positive attributes of micelle polymers which include zero cmc, lower surface activity, low volatility, high electrophoretic mobility, and function as a suitable separation medium even at lower concentrations of pseudophases. In this work, the feasibility of using poly(sodium N-undecanoyl-L-valinate (poly-L-SUV) in CMEKC-MS is demonstrated. After CMEKC separation, enantiomers of 1,1'-binaphthol (BOH) were detected using electrospray ionization mass spectrometry (ESI-MS) by selected ion monitoring (SIM) in the negative ion mode. Although in the SIM mode ESI-MS parameters (nebulizer pressure, drying gas flow rate, drying gas temperature, and sheath liquid flow rate) affected only the signal-to-noise ratio of (+/-)BOH, two of the ESI-MS parameters (nebulizer pressure, sheath flow rate) were found to have a significant impact on chiral resolution of (+/-)BOH. At the optimum ESI-MS conditions, the enantioseparation of (+/-)BOH was successfully accomplished by varying the buffer pH, concentration of the volatile background electrolyte, and poly-L-SUV.     

Comparative glycoproteomics of N-linked complex-type glycoforms containing sialic acid in human serum

This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.     

Sheathless capillary electrophoresis/electrospray ionization mass spectrometry using a pulled bare fused-silica capillary as the electrospray emitter

It has always been assumed that electrical contact at the capillary outlet is a necessary requirement when coupling capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). In this study, we used a pulled bare-capillary tip as the ESI emitter, but neither was it coated with any electrically conductive materials nor was a high external voltage applied on its outlet. In this paper, we demonstrate that this straightforward approach may be used to generate multiply charged ions of proteins and peptides through electrospray ionization. Our results indicate that peptides and proteins, including bradykinin, cytochrome c, myoglobin, and tryptic digest products that elute from a pulled bare-capillary tip can be detected directly by ESI-MS using the tapered bare-capillary interface. Thus, we have demonstrated that CE and ESI-MS may be combined successfully without the need to modify the outlet of the capillary tip with an electrically contacting material.     

Capillary electrophoresis to mass spectrometry interface using a porous junction

A new capillary electrophoresis interface to electrospray ionization mass spectrometry (CE/ESI-MS) is introduced in which the electrical connection to the CE capillary outlet/ESI electrode is achieved by transfer of small ions related to the background electrolyte (BGE) through a porous section near the CE capillary outlet. In this design, only a small section of the capillary wall is made porous. The porous section is created by first thinning a small section of the capillary wall by drilling a well into it and then etching the remaining thin wall porous. This design has two advantages over previous designs (in which the whole circumference of the capillary was made porous): first, the capillary interface is more robust because only a small section of it is made porous, and therefore, no liquid junction is needed to secure the porous section. The electrical connection is achieved simply by inserting the capillary outlet containing the porous junction into the existing ESI needle and filling the needle with the BGE. Second, the time required to make the fused silica porous is reduced from approximately 1 h to a few minutes. In addition, there is no dead volume associated with the porous design, and because the actual metal/liquid contact occurs outside of the CE capillary, bubble formation due to redox reactions of water at the electrode does not affect CE/ESI-MS performance. The performance of this interface is demonstrated by the analyses of peptide and protein mixtures.     

Laser Vaporization/Ionization Interface for Capillary Electrophoresis-Time-of-Flight Mass Spectrometry

Mass spectrometry (MS) is usually coupled on-line with capillary electrophoresis (CE) to analyze biomolecules by using electrospray ionization or continuous-flow fast-atom bombardment. We present a new design for laser vaporization/ionization time-of-flight mass spectrometry. CE, with its low flow rate (<1 μL/min), is highly compatible with MS, even if the total column effluent is introduced directly. A UV laser is used to vaporize and ionize the solution eluting from the column. There is no need to have a makeup solvent. Using this system, we have analyzed a group of amines and peptides. The concentration detection limit of serotonin is in the 10(-)(7) M level. The separation and identification of an amine mixture by CE/MS demonstrates the complementary nature of the information.     

Identification of weak and strong organic acids in atmospheric aerosols by capillary electrophoresis/mass spectrometry and ultra-high-resolution fourier transform ion cyclotron resonance mass spectrometry

A novel approach using a combination of capillary electrophoresis/mass spectrometry (CE/MS) and off-line Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) revealed the structural details of acidic constituents of atmospheric organic aerosol. Both techniques utilized electrospray ionization (ESI), a soft ionization method, to facilitate the analysis of complex mixtures of organic compounds. CE/ESI-MS using an UltraTrol LN-precoated capillary and acidic background electrolytes at different pH values (2.5 and 4.7) was used to differentiate between weak (carboxylic) and strong (sulfonic) organic acids. On the basis of the electrophoretic mobility, m/z constraints from CE/ESI(-)-MS, and elemental composition information retrieved from off-line FTICR-MS, a variety of aliphatic and aromatic carboxylic acids (CHO-bearing molecules), nitrogen-containing carboxylic acids (CHON-bearing molecules), organosulfates (CHOS-bearing molecules), and (nitrooxy)organosulfates (CHONS-bearing molecules) were tentatively identified in the Oasis-HLB-extracted urban PM(2.5) (particulate matter with an aerodynamic diameter of <2.5 μm). The chemical known/unknown structures of detected compounds were confirmed by the semiempirical Offord model (effective mobility linearly correlated to Z/M(2/3)). The majorities of the identified compounds are products of atmospheric reactions and are known contributors to secondary organic aerosols.     

Enhancement of ionization efficiency by electrochemical reaction products in on-line electrochemistry/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

A miniaturized two-electrode electrochemical (EC) cell was developed and was coupled on-line with an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-FTICR MS). Electrochemistry on-line with mass spectrometry, EC/ESI-FTICR MS, of triphenylamine (TPA), which undergoes one-electron oxidation to form a radical cation (TPA*+), demonstrates a significant sensitivity enhancement compared to ESI-FTICR MS. The on-line EC cell configuration with a stainless steel ES needle as the working electrode produces the highest sensitivity in EC/ESI-MS. The results provide evidence that, during the ES ionization, electrolytic reactions occur mainly in the ES tip region, as previously predicted. The results demonstrate that ESI-MS signal suppression by tetrabutylammonium perchlorate electrolyte, which can be a problem, is minimized in EC/ESI-MS. TPA*+ dimer tetraphenylbenzidine (TPB) can be detected by EC/ESI-MS, together with TPA*+, as TPB*+ and TPB2+. The high mass resolving power of FTICR MS was exploited to identify TPB2+ dication in the presence of [TPA*+ - H*]+ ions of the same m/z, from their respective isotopic distributions. The dimer dication TPB2+ can be detected only in EC/ESI-MS.