Chromatographic Behavior Of Peptides Research Articles

Acetic Acid Ion Pairing Additive for Reversed-Phase HPLC Improves Detection Sensitivity in Bottom-up Proteomics Compared to Formic Acid.

Despite the general acceptance of formic acid as the additive of choice for peptide reversed-phase LC-MS/MS applications, some still argue that the selection of acetic acid represents a better option. To settle this debate, we investigated both the difference in MS sensitivity and chromatographic behavior of peptides between these two systems. This interlaboratory study was performed using different MS setups and C18 separation media employing both 0.1% formic and 0.5% acetic acid as ion pairing modifiers. Relative to formic acid, we find an overall ∼2.2-2.5× increase in MS signal and a slight decrease in RP LC retention (-0.7% acetonitrile on average) for acetic acid conditions. While these two features have opposing effects on peptide detectability, we find that acetic acid produces up to 60% higher peptide ID output depending on the type of sample. The drop in RPLC retention increases with peptide net charge at acidic pH. MS signal is dependent on the difference between the charge of the precursor ion and the charge of the peptide in solution, favoring species with a low pI. Lower peptide retention under acetic acid conditions demonstrates its higher hydrophilicity and, as expected, leads to composition and sequence-dependent character of the observed retention shift.

Chromatographic behavior of peptides containing oxidized methionine residues in proteomic LC–MS experiments: Complex tale of a simple modification

On average, the oxidation of a single Met residue to Mso (methionine S-oxide, methionine sulfoxide) and Msn (methionine S,S-dioxide, methionine sulfone) decreases peptide retention in RP HPLC by 2.37 and 1.95 Hydrophobicity Index units (% acetonitrile), respectively. At the same time, the magnitude of the retention shift varies greatly (−9.1 to +0.4% acetonitrile for Mso) depending on peptide sequence. The latter effects are mostly associated with the stabilization of secondary structures upon peptide interaction with the hydrophobic stationary phase: when an oxidized residue is located in the hydrophobic face of an amphipathic helix, the decrease in retention is profound. The same amino acid positioning leads to complete or partial resolution of pairs of peptides containing diastereomeric Mso residues. Contrary to all previously reported observations, and the nature of this modification, we also demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity. This behavior is characteristic for Met residues in the N3 position of an N-capping box stabilization motif prior to the amphipathic helix. All these findings indicate that the prediction of peptide secondary structures upon interaction with hydrophobic surfaces must become an integral part of peptide retention modeling in proteomic applications going forward.

Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry

Because the chromatographic behaviour of peptides is totally different from that of small molecules, a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC–MS is strongly recommended to successfully develop not only qualitative but also quantitative methods. In this study, design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a peptide mixture covering a wide range of molecular weights, isoelectric points and hydropathies. First, a screening design was developed to identify the significant factors concerning mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity (sensitivity). Then, after having selected the experimental domain and the significant factors, a full factorial design was used to further investigate the role of the considered factors and their interactions. Interestingly, ion-pairing reagent nature present in the sample had a tremendous effect on retention and response intensity. Optimal conditions leading to good sensitivity and adequate peptide retention without band splitting were selected and could be used as starting point for rapid method development using classical solvents and ion-pairing reagents.

Mixed-Bed Ion Exchange Chromatography Employing a Salt-Free pH Gradient for Improved Sensitivity and Compatibility in MudPIT

In proteomics, comprehensive analysis of peptides mixtures necessitates multiple dimensions of separation prior to mass spectrometry analysis to reduce sample complexity and increase the dynamic range of analysis. The main goal of this work was to improve the performance of (online) multidimensional protein identification technology (MudPIT) in terms of sensitivity, compatibility and recovery. The method employs weak anion and strong cation mixed-bed ion exchange chromatography (ACE) in the first separation dimension and reversed phase chromatography (RP) in the second separation dimension (Motoyama et.al. Anal. Chem 2007, 79, 3623-34.). We demonstrated that the chromatographic behavior of peptides in ACE chromatography depends on both the WAX/SCX mixing ratio as the ionic strength of the mobile phase system. This property allowed us to replace the conventional salt gradient by a (discontinuous) salt-free, pH gradient. First dimensional separation of peptides was accomplished with mixtures of aqueous formic acid and dimethylsulfoxide with increasing concentrations. The overall performance of this mobile phase system was found comparable to ammonium acetate buffers in application to ACE chromatography, but clearly outperformed strong cation exchange for use in first dimensional peptide separation. The dramatically improved compatibility between (salt-free) ion exchange chromatography and reversed phase chromatography-mass spectrometry allowed us to downscale the dimensions of the RP analytical column down to 25 μm i.d. for an additional 2- to 3-fold improvement in performance compared to current technology. The achieved levels of sensitivity, orthogonality, and compatibility demonstrates the potential of salt-free ACE MudPIT for the ultrasensitive, multidimensional analysis of very modest amounts of sample material.

Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: Implications for comparative proteomics

The differential separation of deuterated and non-deuterated forms of isotopically substituted compounds in chromatography is a well-known but not well-understood phenomenon. This separation is relevant in comparative proteomics, where stable isotopes are used for differential labelling and the effect of isotope resolution on quantitation has been used to disqualify some deuterium labelling methods in favour of heavier isotopes. In this work, a detailed evaluation of the extent of isotopic separation and its impact on quantitation was performed for peptides labelled through dimethylation with H2/D2 formaldehyde. The chromatographic behaviour of 71 labelled peptide pairs from quadruplicate tryptic digests of bovine serum albumin were analysed, focusing on differences in median retention times, resolution, and relative quantitation for each peptide. For 94% of peptides, the retention time difference (heavy-light) was less than 12s with a median value 3.4s. With the exception of a single anomalous pair, isotope resolution was below 0.6 with a median value 0.11. Quantitative assessment indicates that the bias in ratio calculation introduced by retention time shifts is only about 3%, substantially smaller than the variation in ratio measurements themselves. Computational studies on the dipole moments of deuterated labels indicate that these results are consistent with literature suggestions that retention time shifts are inversely related to the polarity of the label. This study suggests that the incorporation of deuterium isotopes through peptide dimethylation at amine residues is a viable route to proteome quantitation.

Effect of cyclization of N-terminal glutamine and carbamidomethyl-cysteine (residues) on the chromatographic behavior of peptides in reversed-phase chromatography

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of −17.03 Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100 Å pore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20 mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10 ≪ trifluoroacetic acid < formic acid. Different approaches for incorporation these findings in the peptide retention prediction models are discussed.

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.

Predictions of peptides' retention times in reversed‐phase liquid chromatography as a new supportive tool to improve protein identification in proteomics

One of the initial steps of proteomic analysis is peptide separation. However, little information from RP-HPLC, employed for peptides separation, is utilized in proteomics. Meanwhile, prediction of the retention time for a given peptide, combined with routine MS/MS data analysis, could help to improve the confidence of peptide identifications. Recently, a number of models has been proposed to characterize quantitatively the structure of a peptide and to predict its gradient RP-HPLC retention at given separation conditions. The chromatographic behavior of peptides has usually been related to their amino acid composition. However, different values of retention coefficients of the same amino acid in different peptides at different neighborhoods were commonly observed. Therefore, specific retention coefficients were derived by regression analysis or by artificial neural networks (ANNs) with the use of a set of peptides retention. In the review, various approaches for peptide elution time prediction in RP-HPLC are presented and critically discussed. The contribution of sequence dependent parameters (e.g., amphipathicity or peptide sequence) and peptide physicochemical descriptors (e.g., hydrophobicity or peptide length) that have been shown to affect the peptide retention time in LC are considered and analyzed. The predictive capability of the retention time prediction models based on quantitative structure-retention relationships (QSRRs) are discussed in details. Advantages and limitations of various retention prediction strategies are identified. It is concluded that proper processing of chromatographic data by statistical learning techniques can result in information of direct use for proteomics, which is otherwise wasted.

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.

On-line strong cation exchange μ-HPLC-ESI-MS/MS for protein identification and process optimization

We have developed an on-line strong cation exchange (SCX)-ESI-MS/MS platform for the rapid identification of proteins contained in mixtures. This platform consists of a SCX precolumn followed by a nanoflow SCX column on-line with an electrospray ion trap mass spectrometer. We also used this platform to study the dynamics of peptide separation/extraction by SCX, in particular to understand the parameters affecting the performance of SCX in multidimensional chromatography. For example, we have demonstrated that the buffer typically used for tryptic digestion of protein mixtures can have a detrimental effect on the chromatographic behaviour of peptides during SCX separations, thereby affecting certain peptide quantitation approaches that rely on reproducible peptide fractionation. We have also demonstrated that band broadening results when a step (discontinuous) gradient approach is used to displace peptides from the SCX precolumn, reducing the separation power of SCX in multidimensional chromatography. In contrast, excellent chromatographic peak shapes are observed when a defined (continuous) gradient is used. Finally, using a tryptic digest of a protein extract derived from human K562 cells, we observed that larger molecular weight peptides are identified using this on-line SCX approach compared to the more conventional reverse phase (RP) LC/MS approach. Both methods used in tandem complement each other and can lead to a greater number of peptide identifications from a given sample.

Prediction of chromatographic retention and protein identification in liquid chromatography/mass spectrometry.

Liquid chromatography coupled on- or off-line with mass spectrometry is rapidly advancing as a tool in proteomics capable of dealing with the inherent complexity in biology and complementing conventional approaches based on two-dimensional gel electrophoresis. Proteins can be identified by proteolytic digestion and peptide mass fingerprinting or by searching databases using short-sequence tags generated by tandem mass spectrometry. This paper shows that information on the chromatographic behavior of peptides can assist protein identification by peptide mass fingerprinting in liquid chromatography/mass spectrometry. This additional information is significant and already available at no extra experimental cost.

A 47-kDa human nuclear protein recognized by antikinetochore autoimmune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation.

Several autoimmune sera from patients with Raynaud phenomenon decorated mammalian kinetochores and bound to a 47-kDa protein on immunoblots of nuclear lysates. Antibody affinity-purified from immunoblots of the 47-kDa band recognized kinetochores, but due to crossreaction with an 18-kDa protein, localization remains elusive. We used one of these sera to purify the antigen from HeLa cells synchronized in mitosis as a noncovalent complex with a 25-kDa protein. The antigen was released from DNA by intercalation with 25 mM chloroquine. Ion-exchange chromatography yielded the pure complex with an apparent molecular size of 68 kDa, which was separated into its components by gel filtration in 6 M guanidinium chloride. Upon two-dimensional gel electrophoresis the 47-kDa protein gave two main spots of pI 6.6 and 6.7, respectively. Posttranslational modification is indicated by additional antigenic spots, by lack of a free alpha-amino group, and by chromatographic behavior of peptides on reversed-phase chromatography. The amino acid sequence for 205 residues of the 47-kDa protein has been established. This sequence is highly homologous with the translated reading frame of RCC1, a gene reportedly involved in regulating onset of mammalian chromosome condensation.