A novel procedure for separating small peptides on polyacrylamide gels

A simple and fast procedure that allows the separation of small(1–3 kDa) peptides on glycine-SDS gels is described. Peptideswere separated by glycine-SDS/PAGE as a result of in situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamidemini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.     

Electrophoretic mobility of Alzheimer's amyloid-beta peptides in urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells.     

Separation of peptides dissolved in a sodium dodecyl sulfate solution by reversed-phase liquid chromatography: removal of sodium dodecyl sulfate from peptides using an ion-exchange precolumn

Separation of peptides by reversed-phase liquid chromatography is significantly affected by sodium dodecyl sulfate (SDS) in the sample solution. The strongly acidic group of SDS binds to the reversed-phase column where it serves as an ion exchanger and retards the elution of peptides. By using a DEAE precolumn connected in series to a reversed-phase column, the interference of SDS in the separation of peptides by reversed-phase chromatography can be significantly diminished. This simple method is applicable to the separation of peptide mixtures obtained by digestion of proteins extracted from SDS-polyacrylamide gels. Peptide production with some proteases in the presence of SDS was examined using the present method. Lysylendopeptidase was suitable for digestion in the presence of SDS, but V8 protease was not.     

H-2 antigens as genetic markers: A two-dimensional gel electrophoretic study

Direct immunoprecipitation and two-dimensional (2D) gel electrophoresis have been used to identify and characterize genetic variation of theH-2K andH-2D regions. Using inbred strains of mice and alloantisera, haplotype-specific polypeptides were defined for five differentH-2 haplotypes. Specific immunoprecipitates prepared from strains of different haplotypes were applied to 2D gels in pairwise combinations to determine whether peptides specific to one haplotype can be distinguished from peptides specific to another. Those haplotype-specific peptides that migrate to unique positions on 2D gels with respect to the positions occupied by haplotype-specific peptides of another haplotype are useful as biochemical genetic markers. Cross-reactivity amongK- andD-region antigens of different haplotypes was identified on 2D gels and found to correlate well with existing data based on serological cross-reactivity. An anti-mouse 2-microglobulin serum was found to be a useful general reagent for immunoprecipitating haplotype-specific H-2 antigens to permit their visualization on 2D gels.Abbrevations used in this paper NP-40 nonidet P-40 - 2D two-dimensional - SDS sodium dodecyl sulfate - IEF isoelectric focusing     

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.     

Comparison of detergent‐based sample preparation workflows for LTQ‐Orbitrap analysis of the Escherichia coli proteome

This work presents a comparative evaluation of several detergent‐based sample preparation workflows for the MS‐based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest‐ and SDS‐based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in‐solution digestion (SC), protein precipitation followed by in‐solution digestion in ammonium bicarbonate or urea buffer, filter‐aided sample preparation (FASP), and 1DE separation followed by in‐gel digestion. On the whole, about 1000 proteins were identified upon LC‐MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.     

Signal peptide amino acid sequences in Escherichia coli contain information related to final protein localization. A multivariate data analysis

With few exceptions, the signal peptides from proteins inserted into, or translocated through, the membranes of gram-negative bacteria or the endoplasmic reticulum of eukaryotes have no sequence homologies. Therefore these signal peptides have not been considered to contain information related to the different final localizations of the proteins. In this study, 43 signal peptide amino acid sequences from proteins with different final localizations in Escherichia coli have been subjected to a multivariate data analysis. Each amino acid residue was characterized by 20 physico-chemical properties, yielding a multivariate property profile for each peptide. The similarities/dissimilarities in the property profiles for the signal peptides from different classes were compared with each other by generating few-dimensional partial least squares (PLS) discriminant plots. With this approach, signal peptides from proteins localized to the periplasmic space (PS), the outer membrane (OM), and the extracellular surroundings (excreted proteins), were separated into distinct groups. Signal peptides from pili proteins were not separated from the OM signal peptides and only partly from the PS signal peptides, but were clearly different from the signal peptides of the excreted proteins. Signal peptides from inner membrane proteins were similar to those of the PS peptides. The size and the hydrophobicity of different peptide segments were responsible for the separation of the signal peptide classes. For example, the hydrophobicity of the N-terminal segment of the signal peptides increased with an increased distance from the cytoplasm of the final localization for the corresponding proteins. Thus, many signal peptides from proteins with different final localizations in E. coli have different discernible physico-chemical profiles.     

Antibacterial mechanisms of GN-2 derived peptides and peptoids against Escherichia coli

Escherichia coli is the main etiological agent of urinary trait infections, able to form biofilms in indwelling devices, resulting in chronic infections which are refractory to antibiotics treatment. In this study, we investigated the antimicrobial and anti-biofilm properties exerted against E. coli ATCC 25922, by a set of peptoids and peptides modeled upon the peptide GN-2, previously reported as a valid antimicrobial agent. The putative antimicrobials were designed to evaluate the effect of cationicity, hydrophobicity and their partitioning on the overall properties against planktonic cells and biofilms as well as on LPS binding, permeabilization of Gram-negative bacteria membranes and hemolysis. The data demonstrated that peptides are stronger antimicrobials than the analogue peptoids which in return have superior anti-biofilm properties. In this study, we present evidence that peptides antimicrobial activity correlates with enhanced LPS binding and hydrophobicity but is not affected by partitioning. The data demonstrated that the enhanced anti-biofilm properties of the peptoids are associated with decreased hydrophobicity and increased penetration of the inner membrane, compared to that of their peptide counterpart, suggesting that the characteristic flexibility of peptoids or their lack of H-bonding donors in their backbone, would play a role in their ability to penetrate bacterial membranes.     

A rapid and easy protein N‐terminal profiling strategy using (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip

Protein N‐terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N‐terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N‐terminal peptide losses. In this study, a fast, one‐step approach utilizing (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N‐terminal peptide enrichment and analysis. Based on the characteristics of TMPP‐derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision‐induced dissociation or HCD fragmentation modes, first the SDS‐PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP‐modified N‐terminal spectra. When utilizing a low amount of starting material (～20 μg protein), a total of 581 yeast N‐terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N‐terminomic studies.     

Separation of lipooligosaccharides by linear gradient gel electrophoresis

Lipooligosaccharides (LOSs) are one of the major antigenic and immunogenic components on the outer membrane of mucosal Gram-negative bacteria. These glycolipid antigens are in the M(r) range of 3-7 kDa, and SDS/PAGE has been used as an analytical tool. Although we are able to separate relatively higher M(r) LOS components by mini-PAGE, we encounter difficulties in resolving LOS components below 3.6 kDa present in heterogeneous LOS preparations. In the present study, we selected PID2 LOS consisting of six LOS components of 3.0-5.1 kDa as a model LOS and examined mini-PAGE conditions not only to resolve smaller M(r) LOS components but also to retain resolving capability of higher LOS components. We found that mini-PAGE with stepwise and linear gradient gels (glycine-SDS) resolved smaller M(r) LOS components. Mini-PAGE with linear gradient gels gave the best resolution, and LOS components of 3.0-5.1 kDa were separated as tight and even bands. Because of the resolution, LOS components were stained chemically and immunochemically much better than those on continuous or stepwise gradient gels. Our study also showed that preformed tricine-SDS (TSDS) minigels such as 16.5 and 10-20% (linear gradient) did not resolve PID2 LOS, which indicated that heterogeneous LOS preparations may not be fully analyzed by using these TSDS minigels. By using glycine-SDS linear gradient mini-PAGE, we should be able not only to screen expression of LOSs but also to characterize smaller M(r) LOS components present in heterogeneous LOS preparations whose identities may have been neglected in the past.     

Capillary electrophoresis of peptides and proteins with plug of Pluronic gel

Electromigration capillary methods are promising techniques in proteomics and they are still under research. We used a partial filling approach, i.e. a combination of gel and non-gel separation mechanisms in a single dimension. We tried using an interesting gel, Pluronic F 127, which can be considered as a surfactant capable of self-association both with isotropic and anisotropic gels. The Pluronic was inserted inside the capillary as a plug at the start of the capillary, and it provided separation at the first time. Separation by this gel was achieved according to molecular weight and/or hydrophobicity. The applicability of this method was demonstrated in the separation of real samples-peptides arising from collagen after CNBr or collagenase cleavage and albumin after trypsin cleavage (peptide mapping). Some peptides and proteins were selectively retained by the Pluronic gel. These interactions with the gel did not depended on their molecular weight alone, but they probably depend on a combination of both principles. It was confirmed that capillary electrophoresis with Pluronic plug can give us another new separation option, complementary to free solution capillary electrophoresis. The CE method presented here, consisting of a partial filling approach with combine gel and non-gel separation mechanisms seemed to be a promising method for the separation of complex mixtures of peptides.     

Competition between functional signal peptides demonstrates variation in affinity for the secretion pathway.

We have developed a system for examining the relative affinity of two different signal peptides for the protein secretion pathway in Escherichia coli. This system involves the expression of a modified alkaline phosphatase which possesses two signal peptides arranged in tandem. When both signal peptides have the wild-type sequence, cleavage after the first and cleavage after the second occur with nearly equal frequency. In both cases the remainder of the protein is transported to the periplasm. Thus both signal peptides effectively compete with each other for entrance to the secretion pathway. When the hydrophobicity of the second signal peptide is altered by small increments, we find that the more hydrophobic signal peptide is preferentially utilized. Thus, a more hydrophobic signal peptide can outcompete even an efficient wild-type signal sequence. The crossover point, for utilization of the second to the first signal peptide, is marked and occurs over a very small change in hydrophobicity. Our results suggest that the small differences in the hydrophobicity of wild-type signal peptides may have critical consequences: preproteins with the more hydrophobic signals could dominate one pathway, leaving those with only slightly less hydrophobic signals to require additional factors such as chaperonins, SecB, and other binding proteins.     

Integrated SDS removal and peptide separation by strong-cation exchange liquid chromatography for SDS-assisted shotgun proteome analysis

We report an improved shotgun method for analyzing proteomic samples containing sodium dodecyl sulfate (SDS). This method is based on the use of strong-cation exchange (SCX) liquid chromatography (LC) for SDS removal that can be integrated with peptide separation as the first dimension of the two-dimensional LC tandem mass spectrometry workflow. To optimize the performance of SDS removal, various experimental conditions, including the concentrations of chemical reagents and salts in the sample, the SDS concentration, and the SCX mobile phase composition, were investigated. It was found that a peptide recovery rate of about 90% could be achieved while removing SDS efficiently. One key finding was that, by increasing the SDS concentration to a certain level (0.5%) in the digested peptide sample, the sample recovery rate could be increased. The peptide recovery rate of BSA digests was found to be 90.6 ± 1.0% (n = 3), and SDS in the SCX fractions collected was not detectable by pyrolysis GC-MS, i.e., below the detection limit of 0.00006% for the undesalted SCX fractions. The peptide recovery rates were found to be 90.9% ± 2.7 (n = 3) and 89.5% ± 0.5% (n = 3) for the digests of the membrane-protein-enriched fractions of E. coli cell lysates and the MCF-7 breast cancer cell line, respectively. Compared to the methods that use acid-labile surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction sample, the SDS method identified, on average (n = 3), more peptides (～5%) and proteins (～16%) than the RapiGest method, while the RapiGest method identified more peptides (～21%) and proteins (～7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods identified more peptides and proteins than the PPS method. Since SCX is widely used as the first dimension of 2D-LC MS/MS, integration of SDS removal with peptide separation in SCX does not add any extra steps to the sample handling process. We demonstrated the application of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein fraction and identified 6889 unique peptides, corresponding to 2258 unique proteins or protein groups from two replicate experiments with a false peptide discovery rate of ～0.8%, compared to 5172 unique peptides and 1847 unique proteins identified by the RapiGest method.     

Characterization and Comparison of Neurofilament Proteins from Rat and Mouse CNS

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co-purifying lower-molecular-weight CNS proteins, including α and β tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co-workers and were demyelinated with Triton X-100. On one-dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower-molecular-weight proteins (63,000-16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two-dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) α and β tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co-purify by the axon flotation procedure also have nonidentical peptide maps; and (4) the corresponding NFPs from rat and mouse have similar peptide maps. The co-purifying proteins examined in detail (63,000–49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.     

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.     

Detection of the mitochondrially encoded cytochrome c oxidase subunit I in the trypanosomatid protozoan Leishmania tarentolae. Evidence for translation of unedited mRNA in the kinetoplast

With the aim of identification of kinetoplast-encoded proteins we investigated the subunit composition of cytochrome c oxidase (respiratory complex IV) from kinetoplast mitochondria of the trypanosomatid protozoan Leishmania tarentolae. Eleven stoichiometric subunits were visible in Coomassie-stained, two-dimensional Blue Native/Tricine-SDS electrophoretic gels. Their partial amino acid sequences indicated that these polypeptides are nuclear-encoded. The mitochondrial subunit I was detected with the polyclonal antibodies against an internal region of this polypeptide. In two-dimensional (9 versus 14%) polyacrylamide glycine-SDS gels this subunit is found as a series of spots located off the main diagonal, a property that can be explained by abnormal electrophoretic migration and aggregation. In gels loaded with high amounts of the purified, enzymatically active oxidase, the subunit I spots could be visualized by staining. The determined N-terminal amino acid sequence of the putative monomeric subunit I (MFXLCLVCLSVS) matched with the predicted sequence, thus indicating that the corresponding kinetoplast unedited mRNA is translated into a functional protein.     

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.     

Estimation of peptide concentration by a modified bicinchoninic acid assay

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 °C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 °C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.     

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides posses strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5–40 μM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt -helical conformation in such anisotropic environments.     

A versatile modelling approach to determine the hydrophobicity of peptides at the atomic level

This study describes a versatile computational method to determine the hydrophobicity of small peptides at the atomic level. Free energies of transfer for individual atoms in peptide structures were derived, utilising two specifically defined parameters: (i) the water-excluding distance to define the dynamic interface between a peptide solute and its surrounding solvent and (ii) the corresponding hydrophobicity index as a relative measure for water occlusion/repulsion. The method was tested on a range of small peptide models (Ac-X-NH₂, G-X-G, Ac-WL-X-LL and Ac-GG-X-GG-NH₂) and several derivatives of these structures, whereby X was any of the 20 most common amino acids that naturally occur in polypeptides or proteins. The advantage of this new method lies in its versatility, ease to implement and capability to provide information on the hydrophobicity characteristics at the atomic level. The approach also encapsulates the impact of factors that influence these properties, but which have hitherto been difficult to accurately quantify, e.g. steric hindrance or proximity effects due to nearby polarised atoms. The method is not conditional on the knowledge of hydrophobicity parameters from the literature and does not require a sophisticated computer software/hardware to enable the atomic solvent-accessible surface areas or other hydrophobicity parameters to be de novo obtained.