Analysis of major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate utilizing capillary electrophoresis mass spectrometry

The separation and detection of the major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate under native conditions has been accomplished for the first time using capillary electrophoresis electrospray ionization-mass spectrometry (CE/ESI-MS). All three major protein-protein and protein-metal complexes in human red blood cells (RBCs) with a concentration dynamic range of approximately 3 orders of magnitude were successfully detected. Intact complexes detected in lysed RBCs included carbonic anhydrase II (CAII-Zn at approximately 0.8 amol/cell) complexed with its zinc cofactor, carbonic anhydrase I (CAI-Zn at approximately 7 amol/cell) complexed with its zinc cofactor, and hemoglobin A (Hb-tetramer at approximately 450 amol/cell)a tetramer formed by two alpha-beta-subunits and four heme groups. The average molecular weights measured for these complexes were consistent with their theoretical values within 0.01% mass accuracy. The use of Polybrene as a self-coating reagent in conjunction with ammonium acetate at pH approximately 7.4, narrow capillary for high separation efficiency, and forward polarity CE to avoid acid production at the tip of the capillary were overriding experimental factors for successful analysis of protein complexes. Diluting the lysed blood sample in ammonium acetate for a minimum of 6 h before injecting the sample into the CE was essential for obtaining the mass accuracy consistent with their theoretical average molecular weights. At physiological pH, the mass spectrum of the electrophoretic peak of Hb-tetramer included a small amount of the monomers and Hb-dimer. The migration time and peak profile of these species were almost identical to that of the tetramer, indicating that they are formed from decomposition of the Hb-tetramer during the ESI process. A separate electrophoretic peak for the Hb-dimer was only detected when the pH of the BGE was lowered from 7.4 to approximately 6.6. Running CE in forward polarity mode was essential for detection of the intact Hb-tetramer as well as CAI-Zn and CAII-Zn complexes. Under forward polarity mode, CE outlet/ESI shared electrode acts as the cathode of the CE circuit and the anode (positive voltage for positive ions) of the ESI circuit, thereby maintaining approximately neutral pH at the CE outlet/ESI electrode. In addition, under forward polarity mode, CAII-Zn and CAI-Zn migrated ahead of Hb-tetramer, avoiding being masked by 562x and 64x, respectively, molar excess of Hb-tetramer.     

Continuous cell introduction for the analysis of individual cells by capillary electrophoresis

Instrumentation for high-throughput analysis of single cells by capillary electrophoresis is described. A flow-based interface that uses electroosmotic flow (EOF) provides continuous injection of intact cells through an introduction capillary into a cell lysis junction and migration of the resulting cell lysate through a separation capillary for analysis. Specifically, two capillaries were coupled together with 5-mm-long Teflon tubing to create a approximately 5-microm gap, and the junction was immersed in a buffer reservoir. High voltage was applied across both capillaries so that cells were continuously pumped into the first capillary by EOF. Individual cells were lysed on-column at the junction without detergents, presumably owing to mechanical disruption caused by a dramatic change in flow properties at the gap. After each cell was lysed at the junction, the major proteins hemoglobin and carbonic anhydrase were separated by capillary electrophoresis and the resultant analyte zones were detected by laser-induced native fluorescence using 275-nm excitation. The detection limits of hemoglobin and carbonic anhydrase were 37 and 1.6 amol, respectively, which correlate well with the literature. The instrumentation was evaluated with intact red blood cells. The averaged time for complete analysis (i.e., continuous injection, lysis, separation, and detection) of one human erythrocyte was less than 4 min with this capillary-based setup. Moreover, this instrumentation simplifies the introduction of individual, intact cells without the use of a microscope.     

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.     

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.     

Fluidic preconcentrator device for capillary electrophoresis of proteins

A new preconcentration device was developed for analysis of proteins by capillary electrophoresis (CE). The microfluidic device uses an electric field to capture proteins that pass through the system. The capture zone is maintained in the flow stream by the interaction between hydrodynamic and electrical forces. The device consists of a flow channel made of PEEK tubing with two electrical junctions, each of which is covered with a conductive membrane. A syringe pump provides the flow stream and also allows the injection of up to 13.5 microL of a dilute sample. The system can be easily connected to a CE device postcapture for off-line preconcentration of proteins. For the proteins used in this study, preconcentration factors up to 40-fold can be achieved. CE detection limits for bovine carbonic anhydrase, alpha-lactalbumin and beta-lactoglobulins A and B were in the nanomolar range using UV detection at 200 nm. Preconcentration is dependent on both time and initial protein concentration. We show the possibility of using an off-line fluidic preconcentrator device employing counterflow capillary electrophoresis with minimum sample manipulation, achieving detection limits similar to on-line approaches.     

Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamide-adenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.     

Fully integrated on-chip electrochemical detection for capillary electrophoresis in a microfabricated device

Microfabricated lab-on-a-chip devices employing a fully integrated electrochemical (EC) detection system have been developed and evaluated. Both capillary electrophoresis (CE) channels and all CE/EC electrodes were incorporated directly onto glass substrates via traditional microfabrication techniques, including photolithographic patterning, wet chemical etching, DC sputtering, and thermal wafer bonding. Unlike analogous CE/EC devices previously reported, no external electrodes were required, and critical electrode characteristics, including size, shape, and placement on the microchip, were established absolutely by the photolithography process. For the model analytes dopamine and catechol, detection limits in the 4-5 microM range (approximately 200 amol injected) were obtained with the Pt EC electrodes employed here, and devices gave stable analytical performance over months of usage.     

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.     

High-Speed TOFMS Detection for Capillary Electrophoresis

A new, high-speed data acquisition system was tested for high storage rate time-of-flight mass spectrometry (TOFMS) detection in capillary electrophoresis (CE). For high spectral acquisition rates of 4 kHz, a spectral storage rate of 80 spectra s(-)(1) was achieved. The resulting detection limit was in the low amol range (10-25 amol) for continuous infusion investigations.     

Separation of recombinant human erythropoietin glycoforms by capillary electrophoresis using volatile electrolytes. Assessment of mass spectrometry for the characterization of erythropoietin glycoforms

The separation of the glycoforms of erythropoietin (EPO) by capillary electrophoresis (CE) was recently published as a monograph by the European Pharmacopoeia (European Pharmacopoeia 4 2002, 1316, 1123-1128). Although the experimental CE conditions employed a background electrolyte containing additives suitable for on-line UV-absorption detection, they were not appropriate for on-line mass spectrometry (MS) detection. In this work, an attempt was made to investigate experimental conditions employing volatile electrolyte systems to achieve the separation and characterization of EPO glycoforms using CE and ESI-MS methodologies. The influence of several operating conditions, such as the coating of the internal walls of the capillary as well as the composition, concentration, and the pH of the separation buffer were investigated. The results demonstrated that when the internal walls of the capillaries were permanently coated with Polybrene and a buffer electrolyte containing 400 mM of HAc-NH4Ac (acetic acid-ammonium acetate), pH 4.75, was used, a significantly reproducible separation was achieved for EPO glycoforms. Intact EPO was characterized by two mass spectrometry techniques: electrospray ionization (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS). The data demonstrated that MALDI-TOF-MS provided a good approximation to an average molecular mass of the EPO molecule. However, it was still necessary to carry out further separation of the intact EPO glycoforms in order to obtain molecular mass information when ESI-MS was used.     

Inline injection microdevice for attomole-scale sanger DNA sequencing

A new affinity-capture-based inline purification, concentration, and injection method is developed for microchip capillary electrophoresis (CE) and used to perform efficient attomole-scale Sanger DNA sequencing separations. The microdevice comprises three axial domains for nanoliter-scale sequencing sample containment, sample plug formation, and high-resolution capillary gel electrophoresis. Purified and concentrated inline sample plugs are formed by electrophoretically driving Sanger sequencing extension fragments into an affinity-capture polymer network positioned within a CE separation channel. Extension fragments selectively hybridize and concentrate at the polymer interface while residual primer, nucleotides, and salts electrophorese out of the system. The plug is thermally released and injected into the CE channel by direct application of the separation voltage. To evaluate this system, 30 nL of sequencing sample prepared from 100 amol (60 million molecules) of human mitochondrial hypervariable region II amplicon was introduced into the microchip, purified, concentrated, and injected, generating a read length of 365 bases with 99% accuracy. This efficient inline injection system obviates the need for the excess sample that is required by cross-injection techniques, thereby enabling Sanger sequencing and other high-performance genetic analysis using DNA quantities approaching theoretical detection limits.     

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.     

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.     

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.     

Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ～6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.     

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.     

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).     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated tapered fused-silica capillary with a beveled edge.

A tapered capillary tip containing a beveled edge was developed for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The optimal flow rate of a 75-microm-i.d., 90-microm-o.d. beveled tapered capillary tip was similar to a conventional flat tapered tip with a 25-microm orifice. Using a mixture of coptisine, berberine, and palmatine chloride, the sheathless CE/ ESI-MS sensitivity of a beveled 75 microm tapered tip capillary was found to be similar to a 25 microm flat tip. Although both tips offer similar CE/ESI-MS sensitivity, the beveled tapered capillary tip is more rugged and durable than a conventional 25-microm tapered capillary because of the larger outside diameter and inside diameter. To make electrical contact, the capillary tip was smeared with paint marker followed by the application of a carbon coating using a graphite pencil. Using this refined carbon-coating procedure, the capillary tip can be operated with aprotic solvents.     

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.     

Attomole-level protein fingerprinting based on intrinsic peptide fluorescence

Protein identification has relied heavily on proteolytic analysis, but current techniques are often slow and generally consume large quantities of valuable protein sample. We report the development of a rapid, ultralow volume protein analysis strategy based on tryptic digestion within the tip of a 1.5-microm capillary channel followed by separation of the proteolytic fragments using capillary electrophoresis (CE). Two-photon excitation is used to probe the intrinsic fluorescence of peptide fragments through "deep-UV" excitation of aromatic amino acid residues at the outlet of the CE channel. Detection limits using this technique are 0.7, 2.4, and 23 amol for the aromatic amino acids tryptophan, tyrosine, and phenylalanine, respectively. In these studies, we demonstrate the capacity to differentiate bovine and yeast cytochrome c variants using less than 15 amol of protein through tryptic fingerprinting. Moreover, the detection of a single amino acid substitution between bovine and canine cytochrome c illustrates the sensitivity of this approach to minor differences in protein sequence. The 2-pL sample volume required for this on-column tryptic digestion is, to our knowledge, the smallest yet reported for a proteolytic assay.