What We See,What We Do Not See,and What We Do Not Want to See in HLA Class I Immunopeptidomes

The identification of peptides bound to human leukocyte antigen class I (HLA‐I) molecules—that is, the HLA‐I immunopeptidome—is a useful tool in the hunt for epitopes suitable for vaccinations and immunotherapies. These peptides are mainly generated by proteasomes through peptide hydrolysis and peptide splicing. In this issue, Nicastri and colleagues compared different methods for the elution of HLA class I‐associated peptides. It is demonstrated that the choice of HLA‐associated peptide enrichment and purification strategy affects peptide yields and creates a bias in detected sequence repertoire. The author carried out this technical brief through the analysis of canonical non‐spliced peptides. However, their study left out any analysis of post‐translationally spliced peptides, thereby missing an opportunity to shed light on the persistent debate of the frequency of these unconventional peptides.     

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide and secretin from the ovine small intestine

Vasoactive intestinal peptide (VIP), peptide histidine isoleucinamide (PHI) and secretin were separated and purified to homogeneity from ovine small intestine, using radioimmunoassay and radioreceptor assay for detection. An efficient and rapid purification sequence included acid extraction, concentration on a bulk C18 cartridge, filtration on a Fractogel column, ion-exchange chromatography on Mono-S and a maximum of three successive reverse-phase HPLC steps. The amounts of peptides obtained from 450 g wet weight tissue were 20 micrograms VIP, 15 micrograms PHI and 5 micrograms secretin. The as yet unknown amino acid sequences of the three peptides were found to be identical to those of the corresponding bovine peptides.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.     

Peptide purification by affinity chromatography based on α‐ketoacyl group chemistry

Significant advances have been achieved in the fields of peptide/protein synthesis, permitting the preparation of large, complex molecules. Shortcomings, however, continue to exist in the area of peptide purification. This paper details some studies we undertook to develop a new strategy for peptide purification based on a reactivity of α‐ketoacyl groups in peptides. The α‐ketoacyl peptide was generated from N^ε‐acyl‐lysyl‐peptide in the solid phase via a transamination reaction using glyoxylic acid and nickel(II) ion. Cleavage of the α‐ketoacyl group with o‐phenylenediamine gave the target peptide in an acceptable yield and purity. We first carried out a careful step‐by‐step optimization of the purification conditions using a model peptide. The strategy was then used in the purification of a transmembrane peptide that could not be effectively purified using a conventional RP‐HPLC system due to the strong hydrophobicity of the peptide and its high tendency to aggregate. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A general strategy for the bacterial expression of amyloidogenic peptides using BCL‐XL‐1/2 fusions

Biophysical studies on amyloidogenic and aggregation‐prone peptides often require large quantities of material. However, solid‐phase synthesis, handling, and purification of peptides often present challenges on these scales. Recombinant expression is an attractive alternative because of its low cost, the ability to isotopically label the peptides, and access to sequences exceeding ～50 residues. However, expression systems that seek to solubilize amyloidogenic peptides suffer from low yields, difficult optimizations, and isolation challenges. We present a general strategy for expressing and isolating amyloidogenic peptides in Escherichia coli by fusion to a polypeptide that drives the expression of attached peptides into bacterial inclusion bodies. This scheme minimizes toxicity during bacterial growth and enables the processing and handling of the peptides in denaturing solutions. Immobilized metal affinity chromatography, reverse phase HPLC, and cyanogen bromide cleavage are used to isolate the peptide, followed by further reverse phase HPLC, which yields milligram quantities of the purified peptide. We demonstrate that driving the peptides into inclusion bodies using fusion to BCL‐XL‐1/2 is a general strategy for their expression and isolation, as exemplified by the production of 11 peptides species.     

High‐sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients’ sera

The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell‐driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA‐bound peptides has typically required billions of cells, still resulting in relatively few high‐confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid‐eluted HLA peptides after purification with the pan‐reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high‐confidence peptides could be identified, including melanoma‐associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology‐based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.     

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS³‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS³ detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E7_11–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.     

The 4‐pyridylmethyl ester as a protecting group for glutamic and aspartic acids: ‘flipping’ peptide charge states for characterization by positive ion mode ESI‐MS

Use of the 4‐pyridylmethyl ester group for side‐chain protection of glutamic acid residues in solid‐phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI‐MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4‐pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI‐MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and purification of amyloidogenic peptides

The polypeptide hormone amylin forms amyloid deposits in patients with type 2 diabetes mellitus. Amyloid-forming peptides are often very difficult to synthesize and purify. Amylin and fragments of amylin are no exception. In this paper we describe the efficient synthesis and purification of two amyloidogenic fragments of human amylin. One fragment corresponds to residues 17 to 37 of the full-length hormone and the other corresponds to residues 24 to 37. These fragments have previously been identified in vivo and have been shown to form amyloid in vitro. The strategy used to elucidate appropriate conditions for the synthesis and purification of these peptides is generally applicable to other peptides that are difficult to synthesize. These peptides were prepared using solid-phase peptide synthesis with Fmoc alpha-amino protection. The effects of varying the solvent, side-chain-protecting group and choice of cleavage conditions were examined. The use of NMP as the main solvent and cleavage with trifluoroacetic acid, phenol, ethanedithiol, thioanisole, and water proved to be optimal. 1,1,1,3, 3,3-Hexafluoro-2-propanol (HFIP) was found to be the best solvent for solubilizing the crude peptides. A wide range of HPLC conditions for the purification of the peptides were examined and an acetonitrile-based solvent system with HCl as the ion pairing agent provided efficient purification.     

Peptide purification using the chemoselective reaction between N‐(methoxy)glycine and isothiocyanato‐functionalized resin

An efficient peptide purification strategy is established, comprising the selective reaction of an N‐terminal N‐(methoxy)glycine residue of the peptide and isothiocyanato‐functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation‐prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240–272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

有机框架材料在多肽富集的应用

生物医药领域近年来发展迅猛,多肽类药物因其生物学活性高、毒性小、生物相容性好等优势,在肿瘤治疗、细胞活性模拟、抗体检测等领域展开广泛应用,多肽的检测分析也成为研究的一大热点。传统的色谱法、溶剂沉淀法、离心超滤法和固相萃取法对多肽能够展现富集效果,但富集的效率往往不够理想。近年来,以有机框架材料为代表的纳米材料,在气体吸附、荧光、传感和催化等领域展开了广泛应用。有机框架材料凭借着独特的结构尺寸,成为了一类理想的生物吸附剂。由于其具有表面可修饰性,能够大大提高对多肽的富集效率。本文着重介绍近5年来金属-有机框架材料（metalorganic frameworks,MOFs）和共价-有机框架（covalent-organic frameworks,COFs）在多肽富集中的应用。     

Peptide enrichment and protein fractionation using selective electrophoresis

In recent times, the analysis of the peptidome has become increasingly valuable to gain a better understanding of the critical roles native peptides play in biological processes. Here, we show a technique using a novel electrophoretic device named MF10, for the fractionation of proteins and peptides based on size and also pH in low volume liquid phase under an electric field. A 1 microM, 7-protein and peptide standard mix ranging from 1 to 25 kDa has been used to show peptide migration into a fraction contained by 1-5 kDa membranes. Simultaneous fractionation of the higher mass protein standards to the correct fraction also occurred. To assess the MF10's ability to fractionate more complex samples, human plasma was used to enrich for the peptidome below 5 kDa in the presence of the proteome. Peptide enrichment was achieved while simultaneously fractionating higher mass proteins to three other mass restricted fractions. The utility of this approach is demonstrated with the identification (with at least 2 ppm mass accuracy) of 76 unique peptides, equating to 22 proteins enriched to the 1-5 kDa fraction of the MF10.     

An Efficient Strategy for Heterologous Expression and Purification of Active Peptide Hainantoxin-IV

Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC₅₀ value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.     

A HLA-DRB supertype chart with potential overlapping peptide binding function

HLA-DRB alleles are class II alleles that are associated with CD4+ T-cell immune response. DRB alleles are polymorphic and currently there are about 622 named in the IMGT/HLA sequence database. Each allele binds short peptides with high sensitivity and specificity. However, it has been suggested that majority of HLA alleles can be covered within few HLA supertypes, where different members of a supertype bind similar peptides showing distinct repertoires. Definition of DRB supertypes using binding data is limited to few (about 29) known alleles (< 5% of all known DRB alleles). Hence, we describe a strategy using structurally defined virtual pockets to group all known DRB alleles with regard to their overlapping peptide binding specificity.     

Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery

Soluble human leukocyte antigen class I (sHLA)‐peptide complexes have been suggested to play a role in the modulation of immune responses and in immune evasion of cancer cells. The set of peptides eluted from sHLA molecules could serve as biomarker for the monitoring of patients with cancer or other conditions. Here, we describe an improved sHLA peptidomics methodology resulting in the identification of 1816 to 2761 unique peptide sequences from triplicate analyses of serum or plasma taken from three healthy donors. More than 90% of the identified peptides were 8–11mers and 74% of these sequences were predicted to bind to cognate HLA alleles, confirming the quality of the resulting immunopeptidomes. In comparison to the HLA peptidome of cultured cells, the plasma‐derived peptides were predicted to have a higher stability in complex with the cognate HLA molecules and mainly derived from proteins of the plasma membrane or from the extracellular space. The sHLA peptidomes can efficiently be characterized by using the new methodology, thus serving as potential source of biomarkers in various pathological conditions.     

Purification of naturally occurring peptides by reversed-phase HPLC

Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (M(r) < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C18), butyl (C4) and diphenyl wide-pore (300 A) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.     

Membrane‐Based SDS Depletion Ahead of Peptide and Protein Analysis by Mass Spectrometry

SDS interferes with both bottom‐up and top‐down MS analysis, requiring removal prior to detection. Filter‐aided sample preparation (FASP) is favored for bottom‐up proteomics (BUP) while acetone precipitation is popular for top‐down proteomics (TDP). We recently demonstrated acetone precipitation in a membrane filter cartridge. Alternatively, our automated electrophoretic device, termed transmembrane electrophoresis (TME), depletes SDS for both TDP and BUP studies. Here TME is compared to these two alternative methods of SDS depletion in both BUP and TDP workflows. To do so, a modified FASP method is described applicable to the SDS purification and recovery of intact proteins, suitable for LC/MS. All three methods reliably deplete >99.8% SDS. TME provide higher sample yields (average 90%) than FASP (55%) or acetone precipitation (57%), translating into higher total protein identifications (973 vs 877 FASP or 890 acetone) and higher spectral matches (2.5 times) per protein. In a top down workflow, each SDS‐depletion method yields high‐quality MS spectra for intact proteins. These results show each of these membrane‐based strategies is capable of depleting SDS with high sample recovery and high spectra quality for both BUP and TDP studies.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

Chemical ligation and cleavage on solid support facilitate recombinant peptide purification

Recombinant peptide technology offers a promising means alternative to chemical synthesis and natural extraction of peptides. The bottleneck in the process of recombinant peptide production is the paucity of efficient purification protocols to eliminate heterogeneity of the desired preparation. Here, we introduce a combination strategy to facilitate purification of recombinant therapeutic peptide via native chemical ligation and chemical cleavage on a solid support. In this study, one promising therapeutic peptide called for type-2 diabetes, GLP-1(7-37), was prepared with high yield and purity without an expensive HPLC purification. Furthermore, this method is also useful for the preparation of isotopically labeled NMR peptide samples. Hopefully, this strategy combining chemical ligation with chemical cleavage on a solid support will ameliorate the production of important recombinant pharmaceutical peptides.