Effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of analytes in liquid chromatographic/ionspray tandem mass spectrometric determination

The effects of liquid chromatography mobile phase buffer contents on the ionization and fragmentation of drug molecules in liquid chromatographic/ionspray tandem mass spectrometric (LC/MS/MS) determination were evaluated for simvastatin (SV) and its hydroxy acid (SVA). The objective was to improve further the sensitivity for SV by overcoming the unfavorable condition caused by the formation of multiple major adduct ions and multiple major fragment ions when using ammonium as LC mobile phase buffer. Mobile phases (70:30 acetonitrile-buffer, 2 mM, pH 4.5) with buffers made from ammonium, hydrazine or alkyl (methyl, ethyl, dimethyl or trimethyl)-substituted ammonium acetate were evaluated. Q1 scan and product ion scan spectra were obtained for SV in each of the mobile phases under optimized conditions. The results showed that, with the alkylammonium buffers, the alkylammonium-adducted SV was observed as the only major molecular ion, while the formation of other adduct ions ([M + H](+), [M + Na](+) and [M + K](+)) was successfully suppressed. On the other hand, product ion spectra with a single major fragment ion were not observed for any of the alkylammonium-adducted SVs. The affinity of the alkylammoniums to SV and the basicity of the alkylamines are believed to be factors influencing the formation and abundance of molecular and fragment ions, respectively. Methylammonium acetate provided the most favorable condition among all the buffers evaluated and improved the sensitivity several-fold for SV in LC/MS/MS quantitation compared with that obtained using ammonium acetate buffer. Better precision for SV in both Q1 and SRM scans was observed when using methylammonium buffer compared with those using ammonium buffer. The mobile phase buffer contents did not seem to affect the ionization, fragmentation and chromatography of SVA. The results of this evaluation can be applied to similar situations with other organic molecules in ionspray LC/MS/MS determination.     

Desorption/ionization mass spectrometry on nanocrystalline titania sol-gel-deposited films

This paper describes a matrix-free method for performing desorption/ionization directly from mesoporous nanocrystalline titania sol-gel thin films, which have good absorption capacity in the ultraviolet (UV) range and can act as assisting materials during UV matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. A high concentration of citrate buffer was added into this system to provide the proton source and to reduce the presence of alkali cation adducts of the analytes. The analyte signals appear uniformly over the whole sample deposition area. Protonated molecules (MH(+) ions) of analytes dominate the titania MALDI mass spectra. Surfactants, peptides, tryptic digest products, and small proteins with molecular weights below ca. 24 000 Da, are observed in the titania MALDI mass spectra. Detection limits for insulin are as low as ca. 2 fmol with mass resolution of ca. 660.     

Importance of matrix: Analyte ratio for buffer tolerance using 2,5-dihydroxybenzoic acid as a matrix in matrix-assisted laser desorption/ionization-fourier transform mass spectrometry and matrix-assisted laser desorption/ionization-time of flight

Many biological samples destined for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) contain buffers. The presence of these buffers often inhibits the ability to obtain spectra. Here, the results of a study of the effects of six different buffers on spectra of three representative small proteins are reported utilizing 2,5-dihydroxybenzoic acid as matrix. These proteins, bovine insulin, cytochrome c, and bovine albumin have masses from ～5000 to 66,000 Da. Three different sample preparation techniques were investigated: aerospray, dried-drop, and acetone redeposition. Both MALDI Fourier transform and time-of-flight mass spectrometry results show that buffer tolerance of MALDI-MS samples depends upon several factors, including the relative amount of the buffer in the MALDI matrix, as well as the identity of the specific buffer. Furthermore, the rate at which buffer tolerance decreases as buffer concentration is increased varies from buffer to buffer. The current results reveal that, at very high matrix:analyte ratios, buffer tolerance of MALDI is dramatically greater than concluded in previous literature reports.     

Phosphate buffers in capillary electrophoresis/mass spectrometry using atmospheric pressure photoionization and electrospray ionization

Capillary electrophoresis (CE) has been combined with atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) for mass spectrometric (MS) detection. Separation conditions using potassium phosphate buffer and ammonium formate buffer have been compared for analysis of eleven pharmaceutical bases. The results showed improvements in separation efficiency and peak symmetry when phosphate buffer was used. The low flow in CE may enable utilization of these advances with MS detection. Compared with ESI, the APPI technique provided a cluster-free background. The enhanced signal-to-noise ratio in the total ion current (TIC) and the reduced spectral background indicated that the APPI process is less affected by non-volatile salts in the CE buffers. This results in a wider range of choice of CE buffers in CE/MS analysis when APPI is the ionization method.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.     

Effect of eluent on the ionization efficiency of flavonoids by ion spray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization mass spectrometry.

The effect of nine different eluent compositions on the ionization efficiency of five flavonoids was studied using ion spray (IS), atmospheric pressure chemical ionization (APCI), and the novel atmospheric pressure photoionization (APPI), in positive and negative ion modes. The eluent composition had a great effect on the ionization efficiency, and the optimal ionization conditions were achieved in positive ion IS and APCI using 0.4% formic acid (pH 2.3) as a buffer, and in negative ion IS and APCI using ammonium acetate buffer adjusted to pH 4.0. For APPI work, the eluent of choice appeared to be a mixture of organic solvent and 5 mM aqueous ammonium acetate. The limits of detection (LODs) were determined in scan mode for the analytes by liquid chromatography/mass spectrometry using IS, APCI and APPI interfaces. The results show that negative ion IS with an eluent system consisting of acidic ammonium acetate buffer provides the best conditions for detection of flavonoids in mass spectrometry mode, their LODs being between 0.8 and 13 microM for an injection volume of 20 microl.     

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states. The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.     

Evaluation of capillary liquid chromatography-electrospray ionization ion mobility spectrometry with mass spectrometry detection

Due to the proteomics revolution, multi-dimensional separation and detection instruments are required to evaluate many peptides and proteins in single samples. In this study, electrospray ionization (ESI) ion mobility spectrometry (IMS) was evaluated as an additional separation after HPLC separations. Common HPLC mobile phase compositions (solvents, acid modifiers, and buffers) were assessed for the effect on ESI-IMS response. Up to 5 mM sodium phosphate, a non-volatile buffer, was able to be electrosprayed into the IMS without degradation of the instrumental performance. Due to the rapid separation times of IMS, multiple IMS spectra were obtained within a single HPLC peak. A five-peptide mixture was separated in a capillary HPLC column under isocratic conditions within 3 min. Coelution of two peaks due to non-optimal HPLC conditions occurred and these two peaks could not be distinguished by HPLC with UV detection. In contrast, the single ion mobility chromatograms provided separation of each peptide as well as providing a second degree of analyte identification (HPLC retention time and IMS mobility). Furthermore, IMS-MS analysis of the five peptides and comparison with HPLC retention times showed that each peptide had a unique retention time-ion mobility-mass to charge value. This work showed that IMS could be employed for direct separation and detection of HPLC eluents and also could be combined with HPLC-MS for three unique dimensions of separation.     

MALDI mass spectrometry analysis of high molecular weight proteins from whole bacterial cells: Pretreatment of samples with surfactants

The use of surfactants as additives in conjunction with on-probe whole cell bacterial protein analysis employing MALDI-TOF-MS is described. Nonionic and zwitterionic surfactants were used to enhance the detection of high molecular weight proteins. Three nonionic, N-octyl-B-D-glactopyranoside, N-decyl-B-D-maltopyranoside, and N-dodecyl-B-D-maltoside, and two zwitterionic surfactants, N,N-dimethyldodecylamine-N-oxide and zwittergent 3-12 were evaluated with five different MALDI matrix systems. New peaks in the mass range of 2 to 80 kDa were produced with all of the various combinations of matrix and surfactant from both whole cell gram-positive and gram-negative bacteria. Ferulic acid used in conjunction with a 1.0 mM solution of N-octyl-B-D-glactopyranoside produced the highest quality spectra with high signal to noise ratios and peaks up to 140 kDa.     

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures

Electrospray mass spectrometry (ES/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) were used to provide mass spectra from seven elapid snake venoms. Spectral interpretation was much simpler for MALDI/TOF/MS. ES/MS proved more useful for the provision of molecular weight data for very closely related peptides, but suppression of higher molecular weight compounds was seen to occur during flow injection analysis. MALDI/TOF/MS proved useful for providing a complete picture of the venom, but the low resolution led to obscuring of major ions, and the mass accuracy was poorer for known peptides. Suppression also occurred during MALDI/TOF/MS but could be overcome using alternative matrices because the spectra were very dependent on the choice of matrix. ES/MS and MALDI/TOF/MS provide complementary and confirmatory information such that for the anal sis of complex peptide mixtures (snake venoms), the use of both techniques is desirable.     

Effect of high-performance liquid chromatography mobile phase components on sensitivity in negative atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

We have investigated the effect of several common buffers (10-mM formic acid, 10-mM ammonium acetate, and 100-mM ammonium acetate) on the ionization of a series of model compounds that are amenable to negative atmospheric pressure chemical ionization to determine the extent of ionization quenching that can occur. In addition, we have compared the sensitivity of these standard mobile phases to a mobile phase that does not contain an acidic buffer component, but rather a base (N-methylmorpholine). The results showed that, as expected, the sensitivity for the test analytes was greatest in the mobile phase that lacked acidic components. In general, ionization of analytes that contained a single, more weakly acidic functional group was inhibited to a greater degree by more strongly acidic buffer components. In some cases, ionization was quenched completely by acidic buffer components, Ionization of compounds that were more strongly acidic was quite good in all mobile phases tested. Differences in the ionization efficiencies of the analytes in each mobile phase were correlated with the gas-phase reagent ions present. As a point of reference, each of the analytes also was analyzed in the positive ion mode and the signal intensities were compared to those obtained in the negative ion mode. In addition, the utility of mobile phases that contained N-methylmorpholine for chromatographic separations was demonstrated.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChip sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrate

The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).     

Accurate mass measurement of DNA oligonucleotide ions using high-resolution time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) time-of-flight mass spectrometry (TOFMS) play an essential role in the analysis of biological molecules, not only peptides and proteins, but also DNA and RNA. Tandem mass spectrometry used for sequence analysis has been a major focus of technological developments in mass spectrometry, but accurate mass measurements by high-resolution TOFMS are equally important. This paper describes the role that high mass measurement accuracy can play in DNA composition assignment and discusses the influence of several parameters on mass measurement accuracy in both MALDI and ESI mass spectra. Five oligonucleotides (5-13mers) were used to test the resolving power and mass measurement accuracy obtained with MALDI and ESI instruments with reflectron TOF mass analyzers. The results from the experimental studies and additional theoretical calculations provide a basis to predict the practical utility of high-resolution TOFMS for the analysis of larger oligonucleotides.     

Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.     

Molecular mass measurement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry

The use of electrospray ionization mass spectrometry for the accurate determination of molecular masses of polynucleotides and small nucleic acids is developed. The common problem of gas phase cation adduction that is particularly prevalent in the mass spectrometric analysis of nucleic acids is reduced through the use of ammonium acetate precipitations and by the addition of chemical additives that compete for adduct ions in solution. The addition of chelating agents such as trans-1,2-diaminocyclohexane-N,N,N,′,N′-tetraacetic acid to remove divalent metal ions and triethylamine to displace monovalent cations from the analyte, in conjunction with ammonium acetate precipitation, reduces cation adduction to levels that permit accurate mass analysis (mass errors of less than 0.01%) without further complex cleanup procedures. The potential utility of accurate mass measurements of small ribonucleic acids is discussed.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection.

On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-microns I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15-20 mM) of ammonium acetate or ammonium formate, and high organic modifier content (5-50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0-0.04 microliter/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous 'infusion' of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.     

X-ray ionization and fragmentation for use with time-of-flight mass spectrometry

In this paper, we introduce laser desorption X-ray ionization for producing ions from the previously undetected neutral species present during laser desorption mass spectrometry. Studies involving the laser desorption of simple sugars were conducted to illustrate the differences between spectra with and without the X-ray source. Ionization was made possible by placing a 200 mCi Am X-ray source directly into the ionization chamber of a time-of-flight mass spectrometer.     

Disappearance of interfering alkali-metal adducted peaks from matrix-assisted laser desorption/ionization mass spectra of peptides with serine addition to alpha-cyano-4-hydroxycinnamic acid matrix

It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H](+) and [M-H](-). The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na](+) and [M+K](+), and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra.     

MALDI post-source decay and LIFT-TOF/TOF investigation of α-cyano-4-hydroxycinnamic acid cluster interferences

Large signals from alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix complexes with sodium and potassium ions were found to interfere with sensitive matrix-assisted laser desorption/ionization (MALDI) analysis of a hydrochloric acid digest of gelatine preparations. The nature of some selected matrix clusters was investigated by conventional post-source decay and LIFT-TOF/TOF experiments. The matrix clusters fragmented readily by neutral evaporation to give smaller sized matrix cluster species without matrix disintegration. Their characterization distinguished them from peptide signals, in particular from those that had the same nominal mass and differed only in the fractional part of the mass as encountered for gelatine-derived peptides. Knowledge of the molecular composition of these cluster species allowed using them for internal calibration of the MALDI mass spectra. The hydrolytic peptides could be analyzed with increased sensitivity when using 2,5-dihydroxy benzoic acid (DHB) as the MALDI matrix.