Peptide retention time prediction for hydrophilic interaction liquid chromatography at acidic pH in formic-acid based eluents.

Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column. Our retention models were able to attain ∼0.98 R2-value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. Overall, the hydrophilicity for these columns decreased when the mobile phase changed pH from 4.5 to 2.7, when using 0.1 % formic acid in the mobile phase. The acidic residues became protonated, and the resultant hydrophilic interaction is dampened at the lower pH, leaving only the basic residues as the primary hydrophilic interactors. Hence, peptides of increasing charge have higher retention. In this comparison between the three columns, ZIC-HILIC has the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, we found that the amino acids at the terminal positions of the peptide modulate the basicity of the N-terminal amino group or the C-terminal Arg/Lys for tryptic peptides. With respect to the separation orthogonality between HILIC and acidic pH RPLC for two dimensional separations, the orthogonality values were lower at pH 2.7 than operating HILIC at pH 4.5 for the first dimension. We also demonstrate that ZIC-HILIC was able to distinguish citrullinated and deamidated peptides based on predicted retention values.

Paradigm Shift: Major Role of Ion-Pairing-Dependent Size Exclusion Effects in Bottom-Up Proteomics Reversed-Phase Peptide Separations.

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Šfully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Špore size materials─was more pronounced for smaller peptides. These findings contradict the central dogmas that underlie the development of all peptide RP-HPLC applications: (i) a larger surface area leads to higher retention and (ii) increasing the pore size should benefit the retention of larger analytes. Based on our intriguing findings, we compared reversed-phase high-performance liquid chromatography peptide retention for a total of 20 columns with pore sizes between 60 and 300 Šusing FA- and TFA-based eluents. Our results unequivocally attest that the larger size of ion pairs in FA- vs TFA-based eluents leads to the observed impact on selectivity and peptide retention. For FA, peptide retention peaks at 200 Špore size, compared to between 120 and 200 Šfor TFA. However, the decrease in retention for narrow-pore particles is more profound in FA. Our findings suggest that common assumptions about analyte size and accessible surface area should be revisited for ion-pair RP separation of small peptides, typical for proteomic applications that are predominantly applying FA eluents. Hybrid silica-based materials with pore sizes of 130-200 Šshould be specifically targeted for bottom-up proteomic applications to obtain both superior peak shape and peptide retentivity. This challenging task of attaining the best RPLC column for proteomics calls for closer collaboration between LC column manufacturers and proteomic LC specialists.

Toward an Ultimate Solution for Peptide Retention Time Prediction: The Effect of Column Temperature on Separation Selectivity.

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of ∼20% of tryptic peptides that were amenable to MS/MS-based identification.

Retention time prediction for post-translationally modified peptides: Ser, Thr, Tyr-phosphorylation.

The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations.

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (ΔHI) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified (y-axis) vs nonmodified (x-axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, pKa of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict ΔHI values.

Deep learning from harmonized peptide libraries enables retention time prediction of diverse post translational modifications.

In proteomics experiments, peptide retention time (RT) is an orthogonal property to fragmentation when assessing detection confidence. Advances in deep learning enable accurate RT prediction for any peptide from sequence alone, including those yet to be experimentally observed. Here we present Chronologer, an open-source software tool for rapid and accurate peptide RT prediction. Using new approaches to harmonize and false-discovery correct across independently collected datasets, Chronologer is built on a massive database with >2.2 million peptides including 10 common post-translational modification (PTM) types. By linking knowledge learned across diverse peptide chemistries, Chronologer predicts RTs with less than two-thirds the error of other deep learning tools. We show how RT for rare PTMs, such as OGlcNAc, can be learned with high accuracy using as few as 10-100 example peptides in newly harmonized datasets. This iteratively updatable workflow enables Chronologer to comprehensively predict RTs for PTM-marked peptides across entire proteomes.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications.

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.

Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments.

We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.1% formic acid) retention upon TMT labeling was found to be 3.3% acetonitrile (linear water/acetonitrile gradients), spanning a range of -4 to 10.3%. In addition to the bulk peptide properties such as length, hydrophobicity, and the number of labeled residues, we found several sequence-dependent features mostly associated with differences in N-terminal chemistry. The behavior of TMTpro-labeled peptides was found to be very similar except for a slightly higher hydrophobicity: an average retention shift of 3.7% acetonitrile. The respective versions of the sequence-specific retention calculator (SSRCalc) model have been developed to accommodate both TMT chemistries, showing identical prediction accuracy (R2 ∼ 0.98) for labeled and nonlabeled peptides. Higher retention for TMT-labeled peptides was observed for high-pH RP and HILIC separations, while SCX selectivity remained virtually unchanged.

Proteomic Shifts Reflecting Oxidative Stress and Reduced Capacity for Protein Synthesis, and Alterations to Mitochondrial Membranes in Neurospora crassa Lacking VDAC.

Voltage-dependent anion-selective channels (VDAC) maintain the bidirectional flow of small metabolites across the mitochondrial outer membrane and participate in the regulation of multiple cellular processes. To understand the roles of VDAC in cellular homeostasis, preliminary proteomic analyses of S100 cytosolic and mitochondria-enriched fractions from a VDAC-less Neurospora crassa strain (ΔPor-1) were performed. In the variant cells, less abundant proteins include subunits of translation initiation factor eIF-2, enzymes in the shikimate pathway leading to precursors of aromatic amino acids, and enzymes involved in sulfate assimilation and in the synthesis of methionine, cysteine, alanine, serine, and threonine. In contrast, some of the more abundant proteins are involved in electron flow, such as the α subunit of the electron transfer flavoprotein and lactate dehydrogenase, which is involved in one pathway leading to pyruvate synthesis. Increased levels of catalase and catalase activity support predicted increased levels of oxidative stress in ΔPor-1 cells, and higher levels of protein disulfide isomerase suggest activation of the unfolded protein response in the endoplasmic reticulum. ΔPor-1 cells are cold-sensitive, which led us to investigate the impact of the absence of VDAC on several mitochondrial membrane characteristics. Mitochondrial membranes in ΔPor-1 are more fluid than those of wild-type cells, the ratio of C18:1 to C18:3n3 acyl chains is reduced, and ergosterol levels are lower. In summary, these initial results indicate that VDAC-less N. crassa cells are characterized by a lower abundance of proteins involved in amino acid and protein synthesis and by increases in some associated with pyruvate metabolism and stress responses. Membrane lipids and hyphal morphology are also impacted by the absence of VDAC.

Peptide retention time prediction for peptides with post-translational modifications: N-terminal (α-amine) and lysine (ε-amine) acetylation.

Development of a peptide retention prediction model in reversed-phase chromatography is reported for acetylated peptides - both N-terminal (α-) and side chain of Lys (ε-amine) residues. Large-scale proteomic 2D LC-MS analyses of acetylated/non-acetylated tryptic digest of whole human cell lysate have been used to assemble representative retention data sets of 25,000+ modified/non-modified pairs. This allowed elucidating chromatographic behaviour of modified peptides in three different separation modes: high pH reversed-phase, HILIC separation on amide phase (first dimension of 2D) and reversed-phase separation with formic acid as ion-pairing modifier in the second dimension. On average, N-terminal acetylation increases peptide RP retention at acidic pH by 5 Hydrophobicity Index units (% acetonitrile). Acetylation of first lysine adds another 4.1%. The magnitude of the retention shift varies greatly depending on the number of modified amines, peptide length, and N-terminal peptide sequence. Large retention shifts have been observed for peptides with hydrophobic N-termini and specifically peptides carrying sequences characteristic for amphipathic helical structures - all in complete agreement with major sequence-specific features of RP retention mechanism. The utility of the modified Sequence Specific Retention Calculator model has been verified for the in-vivo N-terminally acetylated peptides detected by 2D LC-MS/MS analysis of a yeast tryptic digest. The effect of N-terminal acetylation was also evaluated for six different HILIC columns, strong cation- and strong anion exchange separations using previously acquired 2D LC-MS/MS data.

Disrupting Tryptophan in the Central Hydrophobic Region Selectively Mitigates Immunomodulatory Activities of the Innate Defence Regulator Peptide IDR-1002.

Innate defense regulator (IDR) peptides show promise as immunomodulatory therapeutics. However, there is limited understanding of the relationship of IDR peptide sequence and/or structure with its immunomodulatory activity. We previously reported that an IDR peptide, IDR-1002, reduces airway hyperresponsiveness (AHR) and inflammation in a house dust mite (HDM)-challenged murine model of airway inflammation. Here, we examined the sequence-to-function relationship of IDR-1002 in HDM-challenged mice and human bronchial epithelial cells (HBEC). We demonstrated that the tryptophan (W8) in the central hydrophobic region of IDR-1002 is required for the peptide to (i) suppress the pro-inflammatory cytokine IL-33, and induce anti-inflammatory mediators IL-1RA and stanniocalcin-1 in HBEC, and (ii) reduce IL-33 abundance, and eosinophil and neutrophil infiltration, in the lungs of HDM-challenged mice, without affecting the capacity to improve AHR, suggesting multimodal activity in vivo. Findings from this study can be used to design IDR peptides with targeted impact on immunomodulation and pathophysiology in respiratory diseases.

Confident Identification of Citrullination and Carbamylation Assisted by Peptide Retention Time Prediction.

The chromatographic behavior of peptides carrying citrulline and homocitrulline residues in proteomic two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS) experiments has been investigated. The primary goal of this study was to determine the chromatographic conditions that allow differentiating between arginine citrullination and deamidation of asparagine based on retention data, improving the confidence of MS-based identifications. Carbamylation was used as a reference point due to a high degree of similarity between modification products and anticipated changes in chromatographic behavior. We applied 2D LC-MS/MS (a high-pH-low-pH reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC)-low-pH RP, and strong cation exchange (SCX)-low-pH RP) to acquire retention data for modified-nonmodified peptide pairs in the four separation modes. Modifications of a standard protein mixture were induced enzymatically (PAD-2) or chemically (urea) for citrullination and carbamylation, respectively. Deamidation occurs spontaneously. Similar retention shifts were observed for all three modifications in a high-pH RP (decrease) and a low-pH RP (increase), thus limiting the applicability of this 2D LC combination. HILIC on bare silica and strong cation exchange separations have been probed to amplify the effect of charge loss upon citrullination, with SCX demonstrating the most differentiating power: the elimination of basic residues upon citrullination/carbamylation results in an ∼58 mM KCl retention decrease, while retention of deamidated products decreases slightly.

Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions.

Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

Peptide separation selectivity in proteomics LC-MS experiments: Comparison of formic and mixed formic/heptafluorobutyric acids ion-pairing modifiers.

Separation selectivity and detection sensitivity of reversed-phase high-performance liquid chromatography with tandem mass spectrometry analyses were compared for formic (0.1%) and formic/heptafluorobutyric (0.1%/0.005%) acid based eluents using a proteomic data set of ∼12 000 paired peptides. The addition of a small amount of hydrophobic heptafluorobutyric acid ion-pairing modifier increased peptide retention by up to 10% acetonitrile depending on peptide charge, size, and hydrophobicity. Retention increase was greatest for peptides that were short, highly charged, and hydrophilic. There was an ∼3.75-fold reduction in MS signal observed across the whole population of peptides following the addition of heptafluorobutyric acid. This resulted in ∼36% and ∼21% reduction of detected proteins and unique peptides for the whole cell lysate digests, respectively. We also confirmed that the separation selectivity of the formic/heptafluorobutyric acid system was very similar to the commonly used conditions of 0.1% trifluoroacetic acid, and developed a new version of the Sequence-Specific Retention calculator model for the formic/heptafluorobutyric acid system showing the same ∼0.98 R2 -value accuracy as the Sequence-Specific Retention calculator formic acid model. In silico simulation of peptide distribution in separation space showed that the addition of 0.005% heptafluorobutyric acid to the 0.1% formic acid system increased potential proteome coverage by ∼11% of detectable species (tryptic peptides ≥ four amino acids).

Peptide retention time prediction in hydrophilic interaction liquid chromatography: Zwitter-ionic sulfoalkylbetaine and phosphorylcholine stationary phases.

Peptide retention time prediction models have been developed for zwitter-ionic ZIC-HILIC and ZIC-cHILIC stationary phases (pH 4.5 eluents) using proteomics-derived retention datasets of ~30 thousand tryptic peptides each. Overall, hydrophilicity of these stationary phases was found to be similar to the previously studied Amide HILIC phase, but lower compared to bare silicas. Peptide retention is driven by interactions of all charged (hydrophilic) residues at pH 4.5 (Asp, Glu, Arg, Lys, His), but shows specificity according to orientation of functional groups in zwitter-ionic pair. Thus, ZIC-cHILIC exhibits an increased contribution of negatively charged Asp and Glu due to the distal positioning of positively charged quaternary amines on the stationary phase. These findings confirm that HILIC interactions are driven by both peptide distribution between water layer adsorbed on the stationary phase and by interactions specific to functional groups of the packing material. Sequence-Specific Retention Calculator HILIC models were optimized for these columns showing 0.967-0.976 R2-values between experimental and predicted retention values. ZIC-HILIC separations represent a good choice as a first dimension in 2D LC-MS of peptide mixtures with correlations between retention values of ZIC-HILIC against RPLC found at 0.197 (ZIC-HILIC) and 0.137 (ZIC-cHILIC) R2-values, confirming a good orthogonality.

Separation Orthogonality in Liquid Chromatography-Mass Spectrometry for Proteomic Applications: Comparison of 16 Different Two-Dimensional Combinations.

Peptide separation orthogonality for 16 different 2D LC-ESI MS systems has been evaluated. To compare and contrast the behavior of the first dimension columns, a large proteomic retention data set of ∼30 000 tryptic peptides was collected for each 2D pairing. The selection of the first dimension system was made to cover the most popular peptide separation modes applied in proteomics: reversed-phase (RP) separations with different pH, hydrophilic interaction liquid chromatography (HILIC), strong cation and anion exchange (SCX, SAX), and mixed-mode separations. The separation orthogonality generally increases in the order RP < SCX < HILIC < SAX, with the exception of high pH RP-low pH RP system, which showed the second best orthogonality value (68%), just behind PolySAX LP column (74%). The identification output of the 2D LC-MS/MS system is driven by both separation orthogonality and efficiency, making high pH RP the best choice for the first dimension separation. Its performance in combination with a standard C18 at acidic pH can be increased further through the application of pairwise fraction concatenation. The effect of the latter has been evaluated using in silico fraction concatenation, which has been proven to show improvement only for RP separations in the first dimension. Concatenation of two, three, and four-five fractions into one is shown to be the most effective for high pH RP and HFBA- and TFA-based C18 separations, respectively. We also suggest simple guidelines for the unbiased determination of dissimilarity for two separation dimensions and evaluate separation orthogonality in 3D LC-LC-MS separation space for all systems under investigation.

Immunomodulatory Functions of the Human Cathelicidin LL-37 (aa 13-31)-Derived Peptides are Associated with Predicted α-Helical Propensity and Hydrophobic Index.

The anti-endotoxin activity of the cationic peptide LL-37 and its derivative IG-19 is attributed to electrostatic interaction of the peptides' positive charge with negatively charged bacterial lipopolysaccharides (LPS), and in part to the alteration of intracellular mechanisms independent of peptide binding to LPS. We examined the immunomodulatory responses induced by IG-19 and four IG-19-derived scrambled peptides (IG-19a-d), in the presence and absence of LPS, in macrophages and peripheral blood-derived mononuclear cells. All peptides had identical net charge (+5) and amino acid composition, but different hydrophobicity and α-helical propensity. Peptide IG-19 suppressed LPS-induced cytokine/chemokine production by >90%, IG-19a and IG-19b suppressed it by 40-50%, and IG-19c and IG-19d did not suppress cytokine/chemokine production at all. In silico prediction algorithms and the peptide retention time (RT) on a C18 RP HPLC column indicated a linear association between α-helical propensity and hydrophobicity with the ability of the peptides to inhibit LPS-induced responses. Peptide RT exhibited a significant correlation (>70%) between the suppression of LPS-induced cytokine/chemokine production and peptide-induced production of the anti-inflammatory cytokine IL-1RA. These results indicate that RT on a C18 column can be used as a predictor for the immunomodulatory functions of cationic peptides. Overall, we demonstrated that the immunomodulatory functions of LL-37-derived peptides with identical positive charge and amino acid composition are directly associated with the predicted α-helical propensity and hydrophobicity of the peptides.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

Universal retention standard for peptide separations using various modes of high-performance liquid chromatography.

Peptide retention standards are widely used by chromatography specialists. They can be used for quality control of peptide separations (separation efficiency, selectivity, retention values) and for accurate concatenation of retention data from multiple acquisitions in proteomics. So far the repertoire of available retention standards is mostly limited to reversed-phase separations. We introduce a synthetic peptide mixture which can be used in conjunction with the most popular peptide separation techniques: reversed-phase (RPLC), strong-cation exchange (SCX), (strong-anion exchange) SAX and hydrophilic interaction liquid chromatography (HILIC). Target sequences were first designed in-silico using Sequence-Specific Retention Calculator models covering all major peptide separation mechanisms. Peptides were also designed while keeping in mind the simplicity of retention time assignment using MS detection: they all have nearly identical masses and identical intense y3 fragment ions. This contribution demonstrates the application of this mixture for characterization of eight HILIC as well as SAX, SCX and C18 columns.