Biomedical subjects

C G Huber

Publications and source records attributed to C G Huber.

At least 19 recordsLinked to original sources

PubMed2001

Analysis of polymerase chain reaction products by on-line liquid chromatography-mass spectrometry for genotyping of polymorphic short tandem repeat loci.

Capillary ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was used to separate and purify DNA fragments amplified by the polymerase chain reaction (PCR) prior to their characterization by electrospray ionization mass spectrometry (ESI-MS). The investigation by ESI-MS of single- or double stranded species could be effortlessly selected by chromatography of the nucleic acids under either nondenaturing or denaturing conditions, which were realized by proper adjustment of the column temperature. ESI-MS detection sensitivity was improved by a factor of 10 upon replacement of 25 mM triethylammonium bicarbonate as ion-pair reagent by 25 mM butyldimethylammonium bicarbonate because of the applicability of higher acetonitrile concentrations to elute the DNA from the monolithic, poly(styrene/divinylbenzene)-based capillary columns. For fragments ranging in size from 67 to 84 base pairs, the mass accuracies and mass reproducibilities were typically better than 0.02 and 0.008%, respectively, which enabled the characterization and identification of the PCR products with high confidence. The hyphenated method was applied to the genotyping of polymorphic short tandem repeat (STR) loci from the human tyrosine hydroxylase gene (humTH01). The different alleles both in homo- and heterozygotes were identified on the basis of the masses of the single-stranded amplicons and were in full accordance with the alleles identified by conventional capillary electrophoretic sizing.

Alleles↗

PubMed2001

Isoforms of photosystem II antenna proteins in different plant species revealed by liquid chromatography-electrospray ionization mass spectrometry.

The high selectivity offered by reversed-phase high-performance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14-280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.

Chromatography, High Pressure Liquid↗

PubMed2001

Ion chromatography on chelating stationary phases: separation of alkali metals.

Analytical 250x4.6 mm I.D. columns packed with iminodiacetic acid (IDA) derivatised silica were used to separate alkali metal ions and the ammonium ion in combination with non-suppressed conductivity detection. The addition of 2.5-10 mmol/l of the macrocyclic crown ether 18-crown-6 to the nitric acid eluent resulted in a change in the elution order and a significant improvement in the resolution between potassium and ammonium because of selective complexation of potassium. However, the admixture of 15-crown-5 did not improve the resolution of lithium and sodium, although 15-crown-5 is a selective complexing agent for sodium. Retention and resolution of lithium, sodium, ammonium, cesium, rubidium and potassium ions increased at lower temperatures down to 1 degree C. The simultaneous separation of alkali and transition metals under isocratic conditions was achieved with an eluent comprising 10 mmol/l 18-crown-6, 1.5 mmol/l dipicolinic acid, and 1.9 mmol/l nitric acid. The chromatographic system enabled the quantitation of alkali metal ions with detection limits in the low ppb range and excellent linearity. Finally, the applicability of the method was approved by quantitation of sodium, ammonium and potassium in different water samples.

Alkalies↗

PubMed2001

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.

Chromatography, High Pressure Liquid↗

PubMed2001

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.

Alleles↗

PubMed2001

Evaluation of norbornene- beta-cyclodextrin-based monomers and oligomers as chiral selectors by means of nonaqueous capillary electrophoresis.

Norbornen-5-yl carboxylic acid and norbornen-5-ylmethylsilyl ether-based beta-cyclodextrins (beta-CDs) containing up to three norbornene ester and up to five norbornene silyl ether units have been prepared from beta-CD and norbornen-5-carboxylic chloride and norbornen-5-ylmethyldichlorosilane, respectively. Oligomers (n = 2-4) were prepared therefrom using ring-opening metathesis polymerization (ROMP). Monomeric and oligomeric substituted beta-CDs were evaluated as chiral selectors in nonaqueous capillary zone electrophoresis using 35 mM sodium bicarbonate in N-methylformamide (NMF) as background electrolyte. Both monomeric and oligomeric norbornene ester- and norbornene silyl ether-type selectors showed good enantioresolution for dansylated (DNS-) amino acids using concentrations of the chiral selector of up to 4% w/v. A significant improvement in resolution was observed upon the introduction of up to five norbornene silyl ether units into a beta-CD molecule, whereas higher degrees of substitution with norbornen-5-yl-carboxyl groups lead to a reduction in enantioresolution of DNS-amino acids. Thus, pentakis(norbornen-5-ylmethylhydroxysiloxyl)-beta-CD turned out to be superior to mono(norbornen-5-ylmethylhydroxysiloxyl)-beta-CD in terms of enantioresolution. Moreover, norbornene silyl ether-type selectors were found to be more efficient than norbornene ester-type selectors. Finally, oligomeric selectors were found to possess superior or at least comparable enantioselectivity in the separation of DNS-amino acids compared to the parent monomers. A maximum in enantioresolution was obtained with oligo(pentakis(norbornen-5-ylmethylhydroxysiloxyl)beta-CD).

Carbohydrate Sequence↗

PubMed2001

Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometers in electrospray ionization tandem mass spectrometry of oligonucleotides. 1. Comparison of performance characteristics.

The performance of triple-stage quadrupole (TSQ), quadrupole ion trap (QIT), and double focusing sector field (DFSF) mass spectrometers for the generation of fragment ions to obtain sequence information about oligonucleotides was compared. Upon electrospray ionization (ESI), the charge-state distribution of candidate precursor ions not only varied significantly with the type of mass spectrometer, but also with the size and sequence of the investigated oligonucleotides. While concentration limits of detection for an octanucleotide were in the 100 pmol/L range on the QIT and in the 5-10 nmol/L range on the TSQ and DFSF instruments, those of a 24-mer were in the 2-13 nmol/L range on all three instruments. Reproducibility of mass determination, an important prerequisite for reliable identification of fragment ions, was highest on the TSQ with 0.0037% relative standard deviation over three days. Finally, the tandem mass spectra of a dimethoxytritylated pentanucleotide recorded on the three instruments were compared. Relatively simple spectra dominated by complete series of fragment ions of the (a-B) and w type were obtained on the QIT. Complete series of (a-B) and w ions were also observed on the TSQ. However, additional fragments belonging to the b, c, d, x and z series were found in the spectrum. In the spectrum recorded after in-source fragmentation in the DFSF, only fragments corresponding to the loss of a nucleobase and a complete series of w ions were observed. All three mass spectrometers were suitable for the generation of fragment ions, from which the complete nucleotide sequence of the pentanucleotide could be deduced.

Amino Acid Sequence↗

PubMed2001

Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometer in electrospray ionization mass spectrometry. 2. Suitability for de novo sequencing.

The sequence coverage by fragment ions resulting from collision-induced dissociation in a triple stage quadrupole (TSQ) and a quadrupole ion trap (QIT) mass spectrometer of 10-20-mer oligonucleotides was investigated. While (a-B) and w ion series were the most abundant on both instruments, additional ion series of sequence relevance were preferably formed in the TSQ. Thus, a total number of 83 fragment ions were used to deduce the complete sequence of a 10-mer oligonucleotide of mixed sequence from a tandem mass spectrum recorded on the TSQ. The complete sequence was also encoded in the 28 fragments that were obtained from the QIT under comparable fragmentation conditions. Spectrum complexity increased considerably at the cost of signal-to-noise ratio upon fragmentation of a 20-mer oligonucleotide in the TSQ, whereas spectrum interpretation with longer oligonucleotides was significantly more straightforward in spectra recorded on the QIT. The extent of fragmentation had to be optimized by appropriate setting of collision energy and choice of precursor ion charge state in order to obtain full sequence coverage by fragments for de novo sequencing. Moreover, full sequence information was also dependent on base sequence because of the low tendency of backbone cleavage at thymidines. Tandem mass spectrometry on the QIT yielded redundant information that was successfully utilized to deduce the complete sequence of 20-mer oligonucleotides with high confidence.

Base Sequence↗

PubMed2001

Enhancement of complement-mediated lysis by a peptide derived from SCR 13 of complement factor H.

Complement factor H (fH) is an important regulator of complement activation. It contributes to protection of cells against homologous complement attack. In this study we tested the effect of fH-depletion of normal human serum (NHS) on lysis of antibody-coated sheep and human erythrocytes (EshA and EhuA). In the absence of fH, lysis of sensitised Esh and Ehu was clearly increased. Addition of fH to fH-depleted serum re-established protection of cells against complement similar to that seen with NHS. A fH-derived peptide (pepAred), covering the N-terminal half of SCR 13 in fH, was able to enhance complement-mediated lysis of EhuA significantly. However, the oxidised form of this peptide (pepAox) had no effect. Biotinylated pepAred, but not pepAox, was able to directly bind to cells. Additionally, pepAred competed with direct fH-cell interaction which was observable only after treatment of purified fH with mercaptoethanol. Only pepAred increased the amount of C3 fragments and reduced levels of fH detectable on cells as shown by FACS analysis and radio-immuno assay. Furthermore, fH and factor I (fI)-mediated cleavage of agarose bound C3b into iC3b was decreased in the presence of pepAred. These data indicate that a fH-derived peptide can enhance complement-mediated lysis. We will continue to investigate whether the use of a fH peptide can be exploited for therapeutical purposes.

Amino Acid Sequence↗

PubMed2001

Temperature-modulated array high-performance liquid chromatography.

Using novel monolithic poly(styrene-divinylbenzene) capillary columns with an internal diameter of 0.2 mm, we demonstrate for the first time the feasibility of constructing high-performance liquid chromatography arrays for the detection of mutations by heteroduplex analysis under partially denaturing conditions. In one embodiment, such an array can be used to analyze one sample simultaneously at different temperatures to maximize the detection of mutations in DNA fragments containing multiple discrete melting domains. Alternatively, one may inject different samples onto columns kept at the same effective temperature. Further improvements in throughput can be obtained by means of laser-induced fluorescence detection and the differential labeling of samples with up to four different fluorophores. Major advantages of monolithic capillary high-performance liquid chromatographic arrays over their capillary electrophoretic analogs are the chemical inertness of the poly(styrene-divinylbenzene) stationary phase, the physical robustness of the column bed due to its covalent linkage to the inner surface of the fused silica capillary, and the feasibility to modify the stationary phase thereby allowing the separation of compounds not only on the principle of size exclusion, but also adsorption, distribution, and ion exchange. Analyses times are on the order of a few minutes and turnaround time is extremely short as there is no need for the replenishment of the separation matrix between runs.

Chromatography, High Pressure Liquid↗

PubMed2001

Multiplex capillary denaturing high-performance liquid chromatography with laser-induced fluorescence detection.

Denaturing high-performance liquid chromatography (DHPLC) is a sensitive, robust, and operationally inexpensive method for the detection of single-base substitutions and small deletions and insertions. To increase sample throughout, we have developed a multiplexing strategy using fluorophores to distinguish different PCR products. The system combines recent advances in the synthesis of monolithic poly(styrene-divinylbenzene) capillary columns with four-color confocal argon ion laser-induced fluorescence detection. Depending on the change in retention caused by the fluorophores, adjustments in the analysis temperature may be required to ensure the maximum mutation detection sensitivity.

Argon↗

PubMed2000

Preparation and evaluation of packed capillary columns for the separation of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography.

Oligonucleotides and double stranded DNA fragments were separated in 200 microm I.D. capillary columns packed with micropellicular, octadecylated, 2.1 microm poly(styrene-divinylbenzene) particles by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Both the length and the diameter of the connecting capillaries (150 x 0.020 mm I.D.) as well as the detection volume (3 nl) had to be kept to a minimum in order to maintain the high efficiency of this chromatographic separation system with peak widths at half height in the range of a few seconds. Three different types of frits, namely sintered silica particles, sintered octadecylsilica particles, and monolithic poly(styrene-divinylbenzene) (PS-DVB) frits were evaluated with respect to their influence on chromatographic performance. Best performance for the separation of oligonucleotides and long DNA fragments was observed with the PS-DVB frits, whereas the short DNA fragments were optimally resolved in columns terminated by octadecylsilica frits. The maximum loading capacity of 60 x 0.20 mm I.D. columns ranged from 20 fmol (7.7 ng) for a 587 base pair DNA fragment to 500 fmol (2.4 ng) for a 16-mer oligonucleotide. Lower mass- and concentration detection limits in the low femtomol and low nanomol per liter range, respectively, make capillary IP-RP-HPLC with UV absorbance detection highly attractive for the separation and characterization of minute amounts of synthetic oligonucleotides, DNA restriction fragments, and short tandem repeat sequences amplified by polymerase chain reaction.

Base Sequence↗

PubMed2000

High-performance liquid chromatography-electrospray ionization mass spectrometry of single- and double-stranded nucleic acids using monolithic capillary columns.

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200-microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. With gradients of acetonitrile in 100 mM triethylammonium acetate, the synthesized columns allowed the rapid and highly efficient separation of single-stranded oligodeoxynucleotides and double-stranded DNA fragments by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Compared with capillary columns packed with micropellicular, octadecylated poly-(styrene/divinylbenzene) particles, an improvement in column performance of approximately 40% was obtained, enabling the analysis of an 18-mer oligodeoxynucleotide with a column efficiency of more than 190000 plates per meter. The chromatographic separation system was on-line-coupled to electrospray ionization mass spectrometry (ESI-MS). To improve the mass spectrometric detectabilities, 25 mM triethylammonium bicarbonate was utilized as an ion-pair reagent at the cost of only little reduction in separation performance and acetonitrile was added postcolumn as the sheath liquid through the triaxial electrospray probe. High-quality mass spectra of femtomole amounts of 3-mer to 80-mer oligodeoxynucleotides were recorded showing very little cation adduction. Double-stranded DNA fragments ranging in size from 51 to 587 base pairs were separated and detected by IP-RP-HPLC-ESI-MS. Accurate mass determination by deconvolution of the mass spectra was feasible for DNA fragments up to the 267-mer with a molecular mass of 165 019, whereas the spectra of longer fragments were too complex for deconvolution because of incomplete separation due to overloading of the column. Finally, on-line IP-RP-HPLC tandem MS was applied to the sequencing of short oligodeoxynucleotides.

Chromatography, High Pressure Liquid↗

PubMed2000

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection.

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.

Chromatography, Liquid↗

PubMed2000

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry.

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R(F) values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213-216 nm, diode array UV detection, or fluorescence detection (lambda(ex)=330 nm; lambda(cm)=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3',4',5'-trimethoxyflavone. The substances G1-G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC-electrospray ionization tandem mass spectrometry (ESI-MS-MS) of the flavones was carried out employing a 150x2 mm I.D. column packed with a 3 microm/100 A octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water-acetonitrile (50:50). Comparative RP-HPLC-ESI-MS of the raw methanol extract and the isolated substances G1-G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC-ESI-MS-MS was used to identify substances G1-G6 in phytopharmaceutical drugs.

Chromatography, High Pressure Liquid↗

PubMed2000

Sheath liquid effects in capillary high-performance liquid chromatography-electrospray mass spectrometry of oligonucleotides.

Fused-silica capillary columns of 200 microm inner diameter were packed with micropellicular, octadecylated, 2.3 microm poly(styrene-divinylbenzene) particles and applied to the separation of oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography. Oligonucleotides were eluted at 50 degrees C with gradients of 3-13% acetonitrile in 50 mM triethylammonium bicarbonate. Addition of sheath liquid to the column effluent allowed the detection of oligonucleotides by electrospray ionization mass spectrometry using full-scan data acquisition with a detectability comparable to that obtained with UV detection. The signal-to-noise ratios with different sheath liquids increased in the order isopropanol<methanol<acetonitrile. The incorporation of volatile acids or bases such as triethylamine or hexafluoroisopropanol into the sheath liquid was found to influence the charge state distribution of oligonucleotides longer than 20 nucleotide units whereas no significant effect was observed with shorter oligonucleotides. Organic acids and bases in the sheath liquid generally deteriorated the signal-to-noise ratios in the chromatograms and mass spectra mainly because of increased background noise. Only a few charge states were observed in the mass spectra of oligonucleotides because of charge state reduction due to the presence of carbonic acid in the eluent. With triethylammonium hydrogencarbonate as chromatographic eluent and acetonitrile as sheath liquid, very few cation adducts of oligonucleotides were observed in the mass spectra. However, the presence of small amounts of monopotassium adducts enabled the calculation of the charge state of multiply charged ions. With acetonitrile as sheath liquid, 710 amol of a 16-mer oligonucleotide were detected using selected ion monitoring data acquisition with a signal-to-noise ratio of 3:1. Finally, capillary ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was applied to the separation and characterization of heterooligonucleotide primers.

Chromatography, High Pressure Liquid↗

PubMed2000

Comparison of direct infusion and on-line liquid chromatography/electrospray ionization mass spectrometry for the analysis of nucleic acids.

The applicability of ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) and direct infusion/ESI-MS to the characterization of nucleic acid mixtures was evaluated by the analysis of the reaction products obtained from solid-phase synthesis of a 39-mer oligonucleotide. IP-RP-HPLC/ESI-MS was performed using 200 microm i.d. capillary columns packed with octadecylated, micropellicular poly(styrene-divinylbenzene) particles and applying gradients of acetonitrile in 50 mM triethylammonium bicarbonate (TEAB). Three different solvent systems were utilized for direct infusion/ESI-MS with removal of metal cations by on-line cation exchange: (1) 10 mM triethylamine (TEA) in 50% aqueous acetonitrile, (2) 2.2 mM TEA, 400 mM hexafluoro-2-propanol (HFIP) in 20% aqueous methanol and (3) 50 mM TEAB in 10% aqueous acetonitrile. Owing to its separation capability, the highest selectivity and specificity were achieved with IP-RP-HPLC/ESI-MS, which, apart form the 39-mer target sequence, allowed the identification of two isobutyryl-protected target sequences and a 10-mer and 20-mer failure sequence. Direct infusion/ESI-MS with TEA-acetonitrile or TEA-HFIP-methanol as solvent revealed signals for the 39-mer in the m/z range 700-1600. The presence of derivatives containing one, two, three and four isobutyryl groups indicated that the hydrolysis of the protecting groups after solid-phase synthesis was not complete. Failure sequences could not be identified by direct infusion/ESI-MS under conditions favoring multiple charging of the analytes owing to the high chemical background and coincidental overlapping of m/z signals. However, efficient charge state reduction upon addition of carbonic acid to the electrosprayed solvent shifted the signals of the 39-mer and derivatives to m/z values >2400 and allowed the detection of seven different failure sequences, ranging from the 8-mer to the 23-mer, in the mixture.

Base Sequence↗

PubMed1999

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. I. Liquid chromatography.

Proteins ranging in molecular mass from 14,000 to 80,000 were analyzed by reversed-phase high-performance liquid chromatography-electrospray mass spectrometry (RP-HPLC-ESI-MS) using 60 x 1.0 mm I.D. microbore-columns packed with 2.3 microns highly crosslinked, octadecylated poly(styrene-divinylbenzene) particles. Proteins were eluted at temperatures of 80-90 degrees C with gradients of acetonitrile in 0.10-0.50% aqueous solutions of trifluoroacetic acid, formic acid or acetic acid. Substitution of trifluoroacetic acid, the most commonly used mobile phase additive for RP-HPLC, by formic acid resulted in a 35-160-fold improvement in analyte detectability at the cost of an only 32-104% increase in peak width at half height of eluting chromatographic peaks. The lower limits of detection for carbonic anhydrase (M(r) 29,022.7) in full scan and selected ion monitoring mode were 37 and 2.3 fmol, respectively. Measurement of protein masses by RP-HPLC-ESI-MS was accurate and highly reproducible with maximum mass deviations of 0.025% and relative standard deviations of less than 0.011%. Calibration plots of peak area versus concentration allowed the reliable quantitation of proteins in a concentration range of 0.010-1.0 mg/ml. Finally, the optimized method was applied to the separation, identification and quantification of proteins in real samples such as commercial protein preparations, monoclonal antibody fragments, allergen extracts and whey drinks.

Chromatography, High Pressure Liquid↗