The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.     

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

ABSTRACT

Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.     

Characterization of metallothionein isoforms from rabbit liver by liquid chromatography coupled to electrospray mass spectrometry

Metallothioneins (MTs), a group of low molecular weight proteins found in practically all life forms, are characterized by high sulfur content and an affinity for metal ions. At acidic pH, MTs show metal depletion, leading to apothioneins. In the work described here, in order to optimize the separation of rabbit liver apothioneins using liquid chromatography (LC) with UV detection, the proportion of the organic modifier of the mobile phase was optimized by establishing relationships between Reichardt's E(N)(T) scale of solvent polarity and the chromatographic retention measured by the capacity factor, k. Additionally, such optimum separations were carried out in a LC-electrospray mass spectrometry (ES-MS) coupled system allowing the identification and characterization of the different rabbit liver apo-MT-forms. In this way, electrospray ionization mass spectrometry offers great possibilities aiming at a better understanding of metallothionein polymorphism.     

Alternative mobile phases for enhanced chromatographic selectivity and increased sensitivity in peptide separations.

The standard method for separating peptide mixtures is reversed-phase high-performance liquid chromatography with gradients of increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. With modern instruments and columns, complex peptide mixtures can be separated, and low picomole amounts can be collected in tens of microliters. Difficult separations are addressed by modifying the gradient slope or organic eluant composition. Further improvements in resolution are often needed, requiring fundamental changes in mobile phase composition or selection of complementary chromatographic separation mechanisms. For the present study, tryptic digests of cytochrome c from various species were separated in the presence of dilute hydrochloric acid by reversed phase on a Waters Delta-PakTM C18 high-performance insert column and by strong cation exchange on a Waters Protein-PakTM SP 8HR. Different and enhanced reversed-phase selectivity was obtained by replacing trifluoroacetic acid with dilute hydrochloric acid at the same pH. The increased optical clarity of hydrochloric acid-based mobile phases in the low ultraviolet wavelengths yielded increased sensitivity. Very different selectivity was observed with the cation-exchange chromatography. These data expand the options for peptide mapping by providing additional selectivity combined with increased mass sensitivity and spectral information in the low ultraviolet.     

Significance of mobile phase composition in enantioseparation of chiral drugs by HPLC on a cellulose-based chiral stationary phase

Eight randomly selected pharmaceuticals, which included ibuprofen, ketoprofen, albuterol, acebutolol, propafenone, betaxolol, methylphenidate, and homatropine, were directly separated on a cellulose tris(4-methylbenzoate) chiral stationary phase (CSP) without derivatization via normal phase mode HPLC. Enantioresolution was achieved by the optimization of the type and the ratio of mobile phase modifiers and additives. The modifiers included alcohols; the mobile phase additives were trifluoroacetic acid (TFA) and triethylamine (TEA). It was found that methanol and ethanol were superior to isopropanol as mobile phase modifiers for enhancing chiral separation of some of the chiral drugs. The results also demonstrated that TFA has a dominant effect on chiral separations for both acidic and basic chiral drugs, although for some basic drug such as homatropine, TEA was more beneficial at improving enantioseparation. The separation of acebutolol enantiomers was achieved for the first time by adding both TFA and TEA to the mobile phase. The purpose of this paper is to demonstrate that the applicability of cellulose based CSPs can be expanded by controlling the mobile phase compositions through the addition of trace amounts of achiral additives and the selection of the appropriate alcoholic modifier. © 1996 Wiley-Liss, Inc.     

Convenient reduction of S-oxides in synthetic peptides, lipopeptides and peptide libraries

Summary The oxidation of thioether bonds in peptides is still one of the major side reactions during peptide synthesis and it is easily detected by electrospray mass spectrometry. A fast reduction of oxidized methionine, biotin and tripalmitoyl-S-glyceryl-cysteine in synthetic peptides is possible using mixtures of trimethylsilylbromide and ethanedithiol as additives in trifluoroacetic acid. Simple procedures have been worked out which are especially suitable for handling the reduction of large numbers of peptides, prepared by multiple peptide synthesis, and of peptide libraries.     

Parallel SFC/MS-MUX screening to assess enantiomeric purity

Enantiomeric excess (ee) was evaluated for two internally synthesized compound libraries using a high-throughput automated, intelligent four-channel parallel supercritical fluid chromatography/mass spectrometry system equipped with a multiplexed ion source interface (SFC/MS-MUX). The two libraries contained compounds spanning a wide range of enantiomeric ratios with structurally diverse chemical scaffolds and stereogenic centers. The system analyzed each sample simultaneously against four chiral columns using up to six organic modifiers. Enhancements to our previously published parallel supercritical fluid chromatography/mass spectrometry system were implemented to address the challenges associated with automated trace enantiomer identification and quantitation. A reversal of enantiomer elution order was observed for several samples across multiple CSPs and modifiers. The relationship between elution order and % ee accuracy is presented for compounds exhibiting high, middle and low % ee values. Despite incidences in which the minor enantiomer eluted prior to the major enantiomer with less than baseline resolution, the overall % ee was in agreement with separations in which full baseline resolution was achieved. The methods presented here demonstrate the value and utility of high-throughput ee determinations to support drug discovery and development programs.     

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

Mass spectrometric and chemical stability of the Asp-Pro bond in herpes simplex virus epitope peptides compared with X-Pro bonds of related sequences.

The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273-284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography-mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis.     

A rapid and robust liquid chromatography/tandem mass spectrometry method for simultaneous analysis of anti-tuberculosis drugs—Ethambutol and pyrazinamide in human plasma

Ethambutol and pyrazinamide are two first-line anti-tuberculosis drugs. Though they are normally combined for the treatment, their highly different polarity complicates simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of these two drugs in human plasma with decent peak shape and retention. Here we report a rapid and robust LC/MS/MS method for the simultaneous determination of these two drugs in human plasma. Human plasma samples, together with the isotopically labeled internal standards were extracted using protein precipitation, and then separated on a Chromolith SpeedROD RP-18e column and detected with mass spectrometry. The mobile phase is 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol. Addition of trifluoroacetic acid in the mobile phases was found to be able to improve peak shape as well as to increase the retention of ethambutol, thus being able to analyze these two drugs at the same time with both drugs having decent peak shape and enough retention on a C₁₈ column. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The standard curve range was 10.0–5000 ng/mL for ethambutol and 50.0–25,000 ng/mL for pyrazinamide using a plasma sample volume of 50.0 μL. This method has a very short run time of 3.8 min. The method has been fully validated, and <15% relative standard deviation was obtained for both analytes.     

Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.     

A practical dealkylation procedure for O,O-dimethyl-phosphotyrosyl-containing peptide-resins

Summary This paper gives a useful protocol for the demethylation of O,O-dimethyl-phosphotyrosyl peptides on solid support by means of trimethylsilyl iodide in acetonitrile. The method is demonstrated to be well suited for peptide sequences containing arginine, histidine, methionine and tryptophan.Abbreviations DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DIEA diisopropylethylamine - DMA dimethylacetamide - EDT 1,2-ethanedithiol - EGF epidermal growth factor - ESI-MS electrospray ionisation mass spectrometry - Fmoc 9-fluorenylmethoxycarbonyl - HPLC high-performance liquid chromatography - MBHA 4-methyl-benzhydrylamine - NMP N-methyl-2-pyrrolidinone - NMR nuclear magnetic resonance - TFA trifluoroacetic acid - TMSBr trimethylsilyl bromide - TMSCI trimethylsilyl chloride - TMSI trimethylsilyl iodide - TMSOTf trimethylsilyl trifluoromethanesulfonate - TPTU 2-(2-pyridon-1-yl)-1,1,3,3-tetramethyluroniumfluoroborate - t_R retention time Abbreviations for amino acids and nomenclature of peptide structures follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9].     

Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.     

Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC–MS–MS

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI–LC–MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C₁₈ and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column “TFA fix” using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1–36) in rat K₃EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1–34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.     

Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements

The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found.     

High-performance liquid chromatographic enantioseparation of Betti base analogs on a newly developed isopropyl carbamate-cyclofructan6-based chiral stationary phase

The direct separation of the enantiomers of 1-(α-aminoarylmethyl)-2-naphthol, 1-(α-aminoalkyl)-2-naphthol, 2-(α-aminoarylmethyl)-1-naphthol analogs, and 2-(1-amino-2-methylpropyl)-1-naphthol) was performed on a newly developed chiral stationary phase containing isopropyl carbamate-cyclofructan6 as chiral selector, with n-heptane/alcohol/trifluoroacetic acid as mobile phase. The effects of the mobile-phase composition, the nature and concentration of the alcoholic and acidic modifiers, and the structures of the analytes on the retention and resolution were investigated. In some cases, separations were carried out at constant mobile-phase compositions in the temperature range 5-40°C. Thermodynamic parameters and T(iso) values were calculated from plots of ln k' or ln α versus 1/T. -Δ(ΔH°) ranged from 2.8 to 3.2 kJ mol(-1) , -Δ(ΔS°) from 7.7 to 10.1 J mol(-1) K(-1) , and -Δ(ΔG°) from 0.2 to 0.5 kJ mol(-1) . It was found that the enantioseparations were enthalpy driven. The sequence of elution of the stereoisomers determined in some cases was (R) < (S).     

Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides.

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.     

Purification, properties and primary structure of thioredoxin from Aspergillus nidulans.

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (M(r) = 11,564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).     

A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditions

Sample preparation prior to analysis by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) usually involves the storage of frozen peptide samples in an acidic environment for variable time periods. Questions arose in our laboratory regarding the stability of peptides in acid under medium- to long-term storage. Thus, a 10-month longitudinal study was designed to assess the effect on storage of tryptic peptides at −20 and −80 °C under acidic conditions. Our conclusion and proposal from this evaluation is that the optimal storage conditions of peptide samples in acid for proteomic experiments is at −80 °C and, ideally, as separate aliquots.     

Sequencing and model structure of a Naja naja atra protein fragment.

We report the amino acid sequence of a basic protein isolated from the snake venom of Naja naja atra. An automated Edman sequencer was used to determine the 65-residue sequence, aided by electrospray ionization/mass spectrometry. Online reduction and pyridylethylation of the peptide was performed to identify the cysteine residues. Trypsin, chymotrypsin and aspartic digestions were carried out to derive peptide fragments for further sequencing. Fragmented peptides were overlapped to obtain the complete sequence. Molecular mass measurements of the whole protein and its fragments were used as a countercheck for sequence assignment. Further confirmation of the sequence was indicated by sequence homology to other snake venom neurotoxins. A molecular model of the tertiary structure was constructed based on sequence homology, and was refined by global minimization and extensive quality control algorithms. Electrostatic and hydrophobic surface calculations and molecular dynamics simulations were carried out to determine the functional properties of the molecule.