Isomeric Peptides Research Articles

Nano-Antimicrobial Peptides Based on Constitutional Isomerism-Dictated Self-Assembly.

Self-assembly has been identified as an innovative strategy for improving the antimicrobial efficacy and bioavailability of short peptides. However, the detailed molecular information of short peptides linking to the self-assembly structures and antimicrobial activity remains to be more clearly understood. This work reported that the constitutional isomeric sequences of cationic peptides showed a significant impact on their antimicrobial activity. We investigated the self-assembly structures of two constitutional isomeric peptides Ac-RFSFSFR-NH2 and Ac-SFRFRFS-NH2, which contained the same serine, alkaline, and phenylalanine residues but in a different order. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that the constitutional isomers self-assembled into different morphologies in an aqueous solution. The sequence with alkaline residues located at both termini of the peptide favored the formation of β-sheet conformation and nanofibers, while irregular nanospheres were observed when positioning the alkaline residues at the center of the isomeric peptide. The ζ-potential measurements showed that the Ac-RFSFSFR-NH2 nanofibers had a net potential of +17.4 mV, whereas the apparent potential of Ac-SFRFRFS-NH2 nanospheres dropped steeply to +1.0 mV. These differences of the constitutional isomeric peptides were directly reflected in their antimicrobial activities. In comparison with the peptide Ac-SFRFRFS-NH2, the constitutional isomer Ac-RFSFSFR-NH2 exhibited much higher antimicrobial efficacy against Gram-positive Staphylococcus aureus and Bacillus subtilis and Gram-negative Escherichia coli and Pseudomonas aeruginosa. Moreover, several pairs of constitutional isomeric peptides with a similar sequence layout yielded the same outcome. These collective results not only highlight the importance of the isomeric sequence on the antimicrobial efficacy of short peptides but also increase further potential in optimizing the design of self-assembled nano-antimicrobial peptides (AMPs).

Mapping Isomeric Peptides Derived from Biopharmaceuticals Using High-Resolution Ion Mobility Mass Spectrometry

The identification and localization of isomeric peptide modifications is a critical requirement of the biopharmaceutical industry. Despite the ability of liquid chromatography-mass spectrometry to identify many of the common post translational modifications, the identification of isobaric or racemized peptides is confounded by modern mass spectrometry-based techniques. Here, we present a novel approach combining liquid chromatography with a high-resolution ion mobility mass spectrometry system to differentiate peptide and peptide fragments based upon their mobility and mass.

Carbocation Footprinting of Soluble and Transmembrane Proteins

Here, we introduce carbocations (R3C+) as laser-initiated footprinting reagents for proteins. We screened seven candidates and selected trifluomethoxy benzyl bromide (TFBB) as an effective precursor for the electrophilic trifluomethoxy benzyl carbocation (TFB+) under laser (248 nm) irradiation on the fast photochemical oxidation of proteins (FPOP) platform. Initial results demonstrate that this electrophilic cation reagent affords residue coverage of nucleophilic amino acids including H, W, M, and S. Further, the addition of TFB+ increases the hydrophobicity of the peptides so that separation of isomeric peptide products by reversed-phase LC is improved, suggesting opportunities for subresidue footprinting. Comparison of apo- and holo-myoglobin footprints shows that the TFB+ footprinting is sensitive to protein conformational change and solvent accessibility. Interestingly, because the TFB+ is amphiphilic, the reagent can potentially footprint membrane proteins as demonstrated for vitamin K epoxide reductase (VKOR) stabilized in a micelle. Not only does footprinting of the extra-membrane domain occur, but also some footprinting of the hydrophobic transmembrane domain is achieved owing to the interaction of TFB+ with the micelle. Carbocation precursors are stable and amenable for tailoring their properties and those of the incipient carbocation, enabling targeting their soluble or membrane-associated or embedded regions and distinguishing between the extra- and trans-membrane domains of membrane proteins.

A mass spectrometric approach to study the interaction of amyloid β peptides with human α-2-macroglobulin

We used MALDI-MS to study the interaction of amyloid β (Aβ) peptides with alpha-2-macroglobulin (α2M). The binding of amyloid beta (Aβ) peptides to alpha-2-macroglobulin (α2M) was found to inhibit the ability of trypsin to cleave out the peptide α2M 705–715 (Pep-α2M) from α2M. This was observed with both purified α2M and α2M in human serum. We found that Aβ 1–38, Aβ1-40, and Aβ 1–42, all inhibit the interaction of α2M with trypsin, with inhibition rate independent of the length of the Aβ peptide. Further, we show that for complete inhibition, two peptide molecules must be attached to one α2M molecule; one for each of its two subunits. A region was revealed within the Aβ sequence, in which proteolytic cleavage (Lys-28) and oxidation (Met-35) lead to a loss of their ability to inhibit the interaction of trypsin with α2M. Furthermore, we show that after the formation of a trypsin complex with α2M and cleavage of α2M to produce the α2M 705–715, Aβ peptides continue to bind to the protein in the same proportions. However, Aβ peptides treated with DMSO lost their ability to bind to α2M and thereby to inhibit the interaction of trypsin with α2M. While maintaining their primary structure, such an effect can be explained only by conformational changes in the peptides, suggesting the possibility to use our analytical approach to distinguish between conformational isomers of Aβ peptides.

Ion Mobility Spectrometry of Superheated Macromolecules at Electric Fields up to 500 Td.

Since its inception in 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) has been implemented at or near ambient gas pressure. We recently developed FAIMS at 15-30 Torr with mass spectrometry and utilized it to analyze amino acids, isomeric peptides, and protein conformers. The separations broadly mirrored those at atmospheric pressure, save for larger proteins that (as predicted) exhibited dipole alignment at ambient but not low pressure. Here we reduce the pressure down to 4.7 Torr, allowing normalized electric fields up to 543 Td-double the maximum in prior FAIMS or IMS studies of polyatomic ions. Despite the collisional heating to ∼1000 °C at the waveform peaks, the proteins of size from ubiquitin to albumin survived intact. The dissociation of macromolecules in FAIMS appears governed by the average ion temperature over the waveform cycle, unlike the isomerization controlled by the peak temperature. The global separation trends in this "superhot" regime extend those at moderately low pressures, with distinct conformers and no alignment as theorized. Although the scaling of the compensation voltage with the field fell below cubic at lower fields, the resolving power increased and the resolution of different proteins or charge states substantially improved.

Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry.

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra’, which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce ‘3-57 chimera tag’ technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.

Accelerating photofragmentation UV Spectroscopy–Mass spectrometry fingerprinting for quantification of isomeric peptides

Identification of isomeric biomolecules remains a challenging analytical problem. A recently developed spectroscopic method that combines UV photofragmentation and mass spectrometry for fingerprinting of cold ions (2D UV-MS), has already demonstrated its high performance in the library-based identification and quantification of different types of biomolecular isomers. The practical use of the method has been hindered by a slow rate of data acquisition, which makes the fingerprinting incompatible with high-throughput analysis and online liquid chromatography (LC) separation. Herein we demonstrate how the use of a few pre-selected wavelengths can accelerate the method by two orders of magnitude without a significant loss of accuracy. As a proof of principle, 2D UV-MS fingerprinting was coupled to online LC separation and tested for quantification of isomeric peptides containing either Asp or isoAsp residues. The relative concentrations of the peptides mixed in solution have been determined, on average, with better than 4% and 6% accuracy for resolving and non-resolving gradients of LC separation, respectively.

Constitutionally Isomeric Aromatic Tripeptides: Self-Assembly and Metal-Ion-Modulated Transformations.

Self-assembling peptides based on aromatic amino acids can adopt diverse nanostructures which primarily depend on their molecular structures. Therefore, to understand the nature of self-assembly on the molecular level we rationally designed two constitutional isomers of short aromatic peptides. The first isomer consists of a tyrosine moiety at the N-terminus and the second isomer consists of a tyrosine moiety at the C-terminus of the FF peptide, a core recognition motif of Amyloid β peptides. Therefore, it can be considered that both the designed tripeptides are the analogues of the FFF peptide with only atomic(-H) level replacement by -OH functional group on the first and last phenyl ring, respectively. The first isomer self-assembled into 2D porous nanosheets ("Nanowebs"), however the second isomers produced toroidal shapes with central spheres ("Nano-Saturn" like assemblies). Interestingly, the presence of the transition-metal ions (copper, zinc and iron) triggered the self-assembly of both the peptides into fibrous circular discs, nanomats and nanoplates like assembly.

Water-soluble superparamagnetic dysprosium-doped iron oxide flowerlike nanoclusters for high-resolution MR imaging

Magnetic nanomaterials have great application prospects in water treatment, disease diagnosis and therapy, surface adsorption and other fields. Herein, the water-soluble, biocompatible and superparamagnetic dysprosium-doped iron oxide flowerlike nanoclusters (DyIO NCs) were synthesized by thermal decomposition of iron oleate and dysprosium oleate in high-boiling solvent of phenyl ether with α,ω-dicarboxyl poly(ethylene glycol) as a capping agent. The size of the nanoclusters and the amount of Dy ions in the clusters can be controlled by simply adjusting the reaction parameters. The DyIO NCs exhibited excellent colloid stability under physiological conditions. The saturation magnetization and transverse relaxivity (r2) of DyIO NCs were significantly improved by the doping of Dy ions. The r2 value of 21 nm Dy0.209Fe2.791O4 NCs was 485.6 mM−1s−1, which is 9 times that of Dy-doped iron oxide nanoparticles (55.6 mM−1s−1), 4 times that of commercial Feridex (123.6 mM−1s−1) and 2 times that of iron oxide nanoclusters (253.9 mM−1s−1). In vivo imaging showed that DyIO NCs exhibited long blood circulation time and excellent enhancement for T2-weighted magnetic resonance imaging. In addition, the DyIO NCs can be functionalized with tumor targeting ligand cyclic Arg-Gly-Asp peptide and fluorescein isothiocyanate isomer I. This study provides an effective strategy for the development of new sensitive nanoprobes and drug delivery systems, and other applications requiring strong magnetic responses.

High-Resolution Ion Mobility Separations of Isomeric Glycoforms with Variations on the Peptide and Glycan Levels.

Glycosylation is a ubiquitous post-translational modification (PTM) that strongly affects the protein folding and function. Glycosylation patterns are impacted by many diseases, making promising biomarkers. Glycans are also the most complex PTMs, exhibiting isomers (linkage, anomers, and those with isomeric moieties). Permuted with localization variants that occur for all PTMs, these produce numerous isomeric glycoforms. Characterizing them by mass spectrometry and ion mobility spectrometry (IMS) has been a challenge. High-definition differential IMS (FAIMS) had robustly disentangled isomeric peptides involving other PTMs but was not evaluated for glycopeptides that featured multilevel isomerism. Here, we apply it to representative mucin glycopeptides with O-linked glycans: three GalNAc localization variants, a pair with α/β GalNAc anomers, and another with GalNAc/GlcNAc isomers. The first two classes were separated baseline with the resolution exceeding previous benchmarks by 10-fold, and the last pair was partly resolved. The recently demonstrated straightforward coupling to ultrahigh-resolution MS and electron-transfer dissociation makes high-definition FAIMS an attractive tool for glycoproteomics.

Enabling resolution of isomeric peptides using tri-state ion gating and Fourier-transform ion mobility spectrometry

Using Fourier-Transform ion gate modulation technique, we compare the ability of the tri-state ion shutter (3S-IS) to the two-state ion shutter (2S-IS) in separating three pairs of isomeric peptide including 1.Gly-Arg-Gly-Asp-Ser (GRGDS) / Ser-Asp-Gly-Arg-Gly (SDGRG); 2.Sar-Arg-Gly-Asp-Ser-Pro (SRGDSP) / Gly-Arg-Gly-Asp-Thr-Pro (GRGTP); 3.Kemptide / (Val6, Ala7)-Kemptide using electrospray ionization and ion mobility spectrometry. Mobility separation was evaluated for peptide individually and as simple mixtures. Baseline resolution of both singly and doubly charged ions of the isomeric pentapeptide mixture of GRGDS / SDGRG was attainable with the described IMS system using the 3S-IS configuration, illustrating the capacity of the present instrument to resolve isomeric compounds with differences in ion neutral collision cross section (CCS) of less than 1% for the singly charged ions. However, with the 2S-IS, both singly and doubly charged ions of the same peptide mixture were unresolved in the mobility domain. To our knowledge, this is the first-time baseline separation has been reported for the singly charged ions of the isomeric reversed sequence pentapeptide mixture using Fourier transformed drift tube IMS with nitrogen as the drift gas. For all the peptide mixtures, the ion counts for the ion mixture recorded with the 3S-IS were substantially higher (> 50%) in comparison to the 2S-IS. The resolving power of the instrument ranged between 82 to 128 for the target analyte ions analyzed in a mixture using the 3S-IS. Whereas, the resolving power of the 2S-IS ranged between 60 and 100 for the target analytes. Overall, a 20% increase in resolving power was obtained with the 3S-IS in comparison to the 2S-IS. Separation of the different isomeric peptide ion mixture depicted in this present study clearly shows the unique size-to-charge separation ability of IMS that complements the mass-to-charge ratio measurement capacity of mass spectrometry.

Molecular-Level Control over Plasmonic Properties in Silver Nanoparticle/Self-Assembling Peptide Hybrids.

The plasmonic properties of silver nanoparticle (AgNP) arrays are directly controlled by AgNP size, shape, and spatial arrangement. Reported here is a strategy to prepare chiral AgNP arrays templated by two constitutionally isomeric aromatic peptide amphiphiles (APAs), KSC'EKS and C'EKSKS (KS = S-aroylthiooxime-modified lysine, C' = citrulline, and E = glutamic acid). In phosphate buffer, both APAs initially self-assembled into nanoribbons with a similar geometry. However, in the presence of silver ions and poly(sodium 4-styrenesulfonate) (PSSS), one of the nanoribbons (KSC'EKS) turned into nanohelices with a regular twisting pitch, while the other (C'EKSKS) remained as nanoribbons. Both were used as templates for synthesis of arrays of ∼8 nm AgNPs to understand how small changes in molecular structure affect the plasmonic properties of these chiral AgNP/APA hybrids. Both hybrids showed improved colloidal stability compared to pure AgNPs, and both showed enhanced sensitivity as surface-enhanced Raman spectroscopy (SERS) substrates for model analytes, with nanohelices showing better SERS performance compared to their nanoribbon counterparts and pure AgNPs.

Hands-On Electrospray Ionization Mass Spectrometry for Undergraduate Biochemistry Students: Peptide Identification by Ladder Sequencing

Mass spectrometers are ever-increasingly powerful, user-friendly, and affordable. Thus, the addition of mass spectrometry experiments into the undergraduate laboratory curriculum is now both feasible and an effective tool to introduce students to relevant instrumentation. Here an experiment demonstrating the use of a high-resolution electrospray ionization mass spectrometer (ESI-HRMS) for the identification of peptide sequences and the differentiation of isomeric peptides was developed and implemented in a biochemistry laboratory course for third-year chemistry major undergraduate students. This lab activity was a first introduction to mass spectrometry for undergraduate biochemistry students. As a prelab assignment, students were tasked to predict the mass-to-charge ratios and the fragmentation patterns for six given peptides using an online fragmentation prediction tool (Protein Prospector’s MS-Product tool). Students then analyzed two unknown peptides using ESI-HRMS. The theoretical and experimental results were compared to reveal the identity of the two unknown peptides. The student success rate of recognizing the unknown peptide sequences was 87.5%. This laboratory experiment provided students with hands-on experience using a research-grade ESI-HRMS instrument to solve a bioanalytical problem—specifically, the identification of the primary structure of a peptide of unknown sequence. Learning outcomes were evaluated for this experiment, which showed student understanding of peptide sequencing using mass spectrometry.

Age-related isomerization of Asp in human immunoglobulin G kappa chain

Isomerization of aspartate (Asp) is a common non-enzymatic posttranslational modification. Isomerized residues accumulate in proteins associated with age-related human disorders such as cataract and are well known to affect protein structure and function. We previously detected d-Asp-containing peptides in human serum. In this study, we investigated whether isomerized Asp residues are present in human immunoglobulin G (IgG) kappa chain by a qualitative d-amino acid analysis based on diastereomer formation and liquid chromatography tandem mass spectrometry (LC-MS/MS). We also investigated the d/l ratio of Asp residues in the IgG kappa chain in serum from donors aged 25, 37, 41, 54 and 67 years. As a result, two isomerized Asp residues, Asp151 and Asp170, were detected in the IgG kappa chain, and the d/l ratio of these residues was found to increase with aging. To assess the effects of this isomerization, we synthesized four isomeric peptides of IgG kappa chain containing lα-, lβ-, dα-, or dβ-Asp at position 170, and compared their secondary structures by CD spectroscopy. Peptide containing normal lα-Asp170 showed type II β-turn structure, while the other isomeric peptides showed random structure, clearly indicating that substitution of a single Asp isomer alters the secondary structure of the peptide. Because IgG is a main component of humoral immunity, Asp isomerization in IgG may reflect changes of structure and decrease in immune function. Proteome research on serum from the standpoint of racemization might enable us to develop new kinds of biomarker and new directions to study the aging process.

Modulation of Coiled-Coil Binding Strength and Fusogenicity through Peptide Stapling.

Peptide stapling is a technique which has been widely employed to constrain the conformation of peptides. One of the effects of such a constraint can be to modulate the interaction of the peptide with a binding partner. Here, a cysteine bis-alkylation stapling technique was applied to generate structurally isomeric peptide variants of a heterodimeric coiled-coil forming peptide. These stapled variants differed in the position and size of the formed macrocycle. C-terminal stapling showed the most significant changes in peptide structure and stability, with calorimetric binding analysis showing a significant reduction of binding entropy for stapled variants. This entropy reduction was dependent on cross-linker size and was accompanied by a change in binding enthalpy, illustrating the effects of preorganization. The stapled peptide, along with its binding partner, were subsequently employed as fusogens in a liposome model system. An increase in both lipid- and content-mixing was observed for one of the stapled peptide variants: this increased fusogenicity was attributed to increased coiled-coil binding but not to membrane affinity, an interaction theorized to be a primary driving force in this fusion system.

Rapid Quantification of Peptide Oxidation Isomers From Complex Mixtures.

Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here, we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally coelute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis.

Differentiation of peptide isomers by excited-state photodissociation and ion-molecule interactions.

Solvochromatic effects are most frequently associated with solution-phase phenomena. However, in the gas phase, the absence of solvent leads to intramolecular solvation that can be driven by strong forces including hydrogen bonds and ion-dipole interactions. Here we examine whether isomerization of a single residue in a peptide results in structural changes sufficient to shift the absorption of light by an appended chromophore. By carrying out the experiments inside a mass spectrometer, we can easily monitor photodissociation yield as a readout for chromophore excitation. A series of peptides of different lengths, charge states, and position and identity of the isomerized residue were examined by excitation with both 266 and 213 nm light. The results reveal that differences in intramolecular solvation do lead to solvochromatic shifts in many cases. In addition, the primary product following photoexcitation is a radical. Ion-molecule reactions with this radical and adventitious oxygen were monitored and also found to vary as a function of isomeric state. In this case, differences in intramolecular solvation alter the availability of the reactive radical. Overall, the results reveal that small changes in a single amino acid can influence the overall structural ensemble sufficient to alter the efficiency of multiple gas-phase reactions.

Subresidue-Resolution Footprinting of Ligand-Protein Interactions by Carbene Chemistry and Ion Mobility-Mass Spectrometry.

The knowledge of ligand-protein interactions is essential for understanding fundamental biological processes and for the rational design of drugs that target such processes. Carbene footprinting efficiently labels proteinaceous residues and has been used with mass spectrometry (MS) to map ligand-protein interactions. Nevertheless, previous footprinting studies are typically performed at the residue level, and therefore, the resolution may not be high enough to couple with conventional crystallography techniques. Herein we developed a subresidue footprinting strategy based on the discovery that carbene labeling produces subresidue peptide isomers and the intensity changes of these isomers in response to ligand binding can be exploited to delineate ligand-protein topography at the subresidue level. The established workflow combines carbene footprinting, extended liquid chromatographic separation, and ion mobility (IM)-MS for efficient separation and identification of subresidue isomers. Analysis of representative subresidue isomers located within the binding cleft of lysozyme and those produced from an amyloid-β segment have both uncovered structural information heretofore unavailable by residue-level footprinting. Lastly, a "real-world" application shows that the reactivity changes of subresidue isomers at Phe399 can identify the interactive nuances between estrogen-related receptor α, a potential drug target for cancer and metabolic diseases, with its three ligands. These findings have significant implications for drug design. Taken together, we envision the subresidue-level resolution enabled by IM-MS-coupled carbene footprinting can bridge the gap between structural MS and the more-established biophysical tools and ultimately facilitate diverse applications for fundamental research and pharmaceutical development.

Relative Quantitation of Beta-Amyloid Peptide Isomers with Simultaneous Isomerization of Multiple Aspartic Acid Residues by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry can be used for rapid quantitation of peptides with various post-translational modifications (PTM), even if they do not shift the mass of the native peptide. Previously, it was shown that MALDI-TOF MS can be used for quantitation of isoD7 beta-amyloid 1-42 peptide. On the basis of the differences in the collision-induced dissociation fragmentation pattern of native Aβ, isoD7 Aβ, isoD23 Aβ, and isoD7_23 peptide (a di-isomerized peptide with both isomerization of D7 and D23 residues), we developed a MALDI-TOF-based method for simultaneous quantitation of all of these isoforms. Using multivariate regression for analysis of fragment MS data, the method allows the determination of the molar fractions of all of these isoforms with up to 16% error for mixtures with 2 pmol total amount of the beta-amyloid peptide.

A peculiar chromatographic selectivity of porous graphitic carbon during the separation of dileucine isomers

Porous graphitic carbon is a versatile stationary phase for high-performance liquid chromatography which performs especially well for isomeric separations. Shape-sensitivity of the stationary phase is caused by a steric effect when a molecule interacts with a flat carbon surface. It follows that branched, non-flat molecules are eluted much earlier than flat or linear molecules. In this short communication we show that if a molecule has a highly ionizable group, the “shape” of a molecule part which is farther from the ionizable group affects retention much more than the “shape” of a molecule part which is closer to the ionizable group. Dipeptides which consist of tert-leucine and norleucine were used as an example. Basic and acidic eluents were used. Retention strongly depends on whether a norleucine or tert-leucine residual is located near the non-ionized side in an eluent for both basic and acidic eluents. A residual located on the opposite side is less important. To investigate the possible causes of this peculiar retention behavior we compared the retention behavior of these dipeptides for porous graphitic carbon with the behavior for other types of stationary phases and with the calculated physicochemical properties. Strong and complex dependence of elution order on a mobile phase composition is demonstrated. The separation of other dileucine isomers is also considered. The applicability of porous graphitic carbon for the separation of complex mixtures of isomeric peptides is discussed.