Separation Of Peptide Mixtures Research Articles

Protein profiling and phosphoprotein analysis by isoelectric focusing.

Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification.

Optimal selection of 2D reversed-phase–reversed-phase HPLC separation techniques in bottom-up proteomics

Evaluation of: Stephanowitz H, Lange S, Lang D et al. Improved two-dimensional reversed phase–reversed phase LC-MS/MS approach for identification of peptide–protein interactions. J. Proteome Res. 11(2), 1175–1183 (2011).Recent developments in bottom-up proteomics have supplanted the use of gel-based approaches in favor of multidimensional chromatographic separations of peptide mixtures followed by mass spectrometry analysis. This trend is driven by the desire to eliminate labor-intensive in-gel digestion procedures and increase proteome coverage through better recovery of proteolytic fragments. Introduction of reversed-phase–reversed-phase 2D separation techniques is one of the major improvements that have made this possible. In this article, we review recent developments in 2D HPLC and highlight variations in reversed-phase HPLC separation selectivity that allow for superior peak capacity in peptide fractionation.

Design of peptide standards with the same composition and minimal sequence variation to monitor performance/selectivity of reversed-phase matrices

The present manuscript extends our de novo peptide design approach to the synthesis and evaluation of a new generation of reversed-phase HPLC peptide standards with the same composition and minimal sequence variation (SCMSV). Thus, we have designed and synthesized four series of peptide standards with the sequences Gly-X-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys-amide, where the N-terminal is either Nα-acetylated (Series 1) or contains a free α-amino group (Series 3); and Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal is either Nα-acetylated (Series 2) or contains a free α-amino group (Series 4). In this initial study, the single substitution position, X, was substituted with alkyl side-chains (Ala<Val<Ile, in order of increasing hydrophobicity) or aromatic side-chains (Phe, Tyr). Peptide series pairs 1/2 and 3/4 thus represent SCMSV peptides, with the substitution site, X, being towards the N- or C-terminal, albeit with identical adjacent residues (Gly-X-Leu) to maintain the same environment around position X. In addition, peptide series pairs 1/3 and 2/4 enable an examination of the effect of a free, positively charged α-amino group on peptide retention behaviour relative to a blocked N-terminus. Peptide mixtures were run at pH 2 on columns with a variety of stationary phase selectivity (C8, C18, polar endcapped, polar embedded, ether-linked phenyl and Phenyl-Hexyl) under linear gradient conditions with acetonitrile or methanol as organic modifier. It was interesting to note that the addition of the hydroxyl group to the aromatic ring in a 12-residue Tyr SCMSV peptide pair had a dramatic effect on resolution compared to the Phe peptide pair. In addition, SCMSV peptide pairs with the β-branched Val and Ile side-chains at position X were the most difficult to separate compared to SCMSV peptides containing the aromatic side-chains Tyr and Phe. In this initial study, SCMSV peptide pairs proved to be a potent test of the selectivity of reversed-phase packing materials. In addition, mixtures of SCMSV peptide standards to assess overall capabilities of stationary phases to resolve complex peptide mixtures underlined the useful complementarity of combinations of different columns and elution conditions to maximize flexibility in peptide applications. Finally, our controlled, de novo designed peptide approach should spur the development of more quantitative selectivity parameters for peptide separations, such as those already available for small molecules, enhancing further the universal value of utilizing peptide standards to compare column performances in the separation of peptide mixtures.

Optimization of conditions for the separation of peptide mixtures using strong cation exchange chromatography

The separation conditions for strong cation exchange chromatography were investigated and compared using tryptic digest of protein extract from yeast. The conditions included the loading amount of the sample, the type of the salt in mobile phase, the type of the acid used to adjust the pH of the mobile phase and the proportion of organic solvent added in the elution solution. The experimental results indicated that high separation efficiency can be obtained by using ammonium chloride solution as optimal mobile phase, phosphoric acid to adjust the pH of mobile phases and 30% (v/v) organic solvent in mobile phases. The results suggested that a beneficial reference was presented for the selection of separation conditions of strong cation exchange chromatography when applying in two-dimensional chromatography-mass spectrometry strategy in proteomic research.

The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry

To investigate the proteome composition and function of human neonatal arterial umbilical cord. Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS). A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel biomarkers, such as alpha-fetoprotein and periostin that were specific to newborn infants. These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.

The tear proteomics analysis of conjunctivochalasis

To analysis tear proteins by using shotgun strategy, and to study pathogenesis mechanism of conjunctivochalasis by comparing tear proteins between the conjunctivochalasis and normal body. Tears were obtained from 8 conjunctivochalasis cases and 8 normal controls. Fifteen microliters of tears was collected by microcapillary tubes from each eye. Shotgun strategy was used for tear protein analysis. Trypsin digestion in-solution, separation of peptide mixture by reverse-phase high-pressure liquid chromatograph (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS/MS) identification and bioinformation analysis were used in this study. Three hundred and fifty-six proteins were excised in conjunctivochalasis patients and 352 proteins in the normal controls, and among them 119 proteins were the same. The functions of tear proteins were classified with GOA, which found some apoptosis regulation proteins and apoptosis related proteins and inflammatory response related proteins in conjuntivochalasis but not in normal controls group. Defensin was also found in conjuntivochalasis. Shotgun strategy can separate and analysis tear proteins effectively, which provide a new method for tear protein component of conjunctivochalasis. The special component in conjunctivochalasis tear show conjunctivochalasis maybe related to cell apoptosis and inflammatory.

Use of Proteomics Analysis for Molecular Precision Approaches in Cancer Therapy

The rapidly expanding data sets derived from genomic and transcriptomic analyses have allowed greater understanding of structural and functional network patterns within the genome resulting in a realignment of thinking within a systems biologic framework of cancer. However, insofar as spatially and temporally dynamic differential gene expression at the protein level is the mediate effector of cellular behavior and, in view of extensive post translational modification (PTM), the need for sensitive, quantitative, and high throughput proteomic analytic techniques has emerged. To circumvent the problems of tissue sample heterogeneity, laser capture microdissection (LCM) allows for the acquisition of homogeneous cell populations. Using different fluorescent dyes to label protein samples prior to gel electrophoresis, 2-D DIGE (twodimensional differential in-gel electrophoresis) can, with reasonable sensitivity, process three protein samples on the same gel allowing for intragel relative quantification. MudPIT (multidimensional protein identification technology) is a non-gel approach exploiting the unique physical properties of charge and hydrophobicity which allows the separation of peptide mixtures as well as direct MS (mass spectrometry) and database searching. The introduction of iTRAQ (isobaric tags for relative and absolute quantification) achieves labeling of all peptides by employing an 8-plex set of amine reactive tags to derivatize peptides at the N-terminus and lysine side chains allowing for absolute quantification and assessment of PTM. These and other new laboratory technologies, along with improved bioinformatics tools, have started to make significant contributions in cancer diagnostics and treatments.

Novel chromatography techniques for high-throughput analysis of proteomes

Due to their rather high complexity, most analyses of proteome samples have been accomplished by multidimensional separation techniques based on either top-down or bottom-up strategies. In the former strategy, all proteins extracted from a biological sample are separated by twodimensional gel electrophoresis (2D-GE)—introduced by O’Farrell in 1975 [1]—in which proteins are separated on a flat gel according to their isoelectric points (pIs) by isoelectric focusing (IEF) on the first dimension and molecular weights (MWs) by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) on the second dimension. Because of its unparalleled resolving power, 2D-GE has become an important technique in proteomic analysis [2]. However, it is a manual, time-consuming technique with poor sensitivity, poor quantifying power and a limited dynamic range. Moreover, it is difficult to investigate rather hydrophobic/hydrophilic proteins and those that occur with low copy number in whole cell lysate using this technique. All of these drawbacks have undermined its prospects of being the dominant separation technique in proteome research in the future. As an alternative approach, the bottom-up strategy has been paid increasing attention over the past decade. In this approach, the digest of all extracted proteins is first separated by two-dimensional high-performance liquid chromatography (2D-HPLC), and then the components are identified by tandem mass spectrometry (MS/MS) [3]. More recently, Yates and his colleagues developed a multidimensional protein identification technology (MUDPIT) that involves the 2D-HPLC separation of peptide mixtures using an orthogonal biphasic capillary column packed with strong cation exchange (SCX) resin and reversed-phase (RP) materials in tandem. They applied this novel technology to the analysis of the whole yeast proteome, and 5440 peptides (corresponding to 1484 unique proteins) were identified, among which a substantial number of low-abundance and trans-membrane proteins were identified [4]. However, the greatest impediment to the widespread use of 2D-HPLC platforms is currently the rather long analysis time required—generally about up to days per analysis. Therefore, it is crucial to accelerate the whole analysis procedure. In this article, recent advances in the development of novel chromatography techniques for the high-throughput analysis of proteomes are summarized.

Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5'-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5'-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

The human plasma proteome: Analysis of Chinese serum using shotgun strategy

We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20 healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1) removal of six high-abundant proteins; (2) tryptic digestion of low- and high-abundant proteins of serum; (3) separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944 nonredundant proteins were identified under a stringent filter condition (X(corr) > or = 1.9, > or = 2.2, and > or = 3.75, < or = C(n) > or = 0.1, and R(sp) > or = 4.0) in both pooled male and female samples, in which 594 and 622 entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206 proteins were identified with at least two distinct peptides per protein and 185 proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery.

Device for the reversed-phase separation and on-target deposition of peptides incorporating a hydrophobic sample barrier for matrix-assisted laser desorption/ionization mass spectrometry.

The separation of peptide mixtures from proteolytic cleavage is often necessary prior to mass spectrometry (MS) to enhance sensitivity and peptide mapping coverage. When buffers, salts, and other higher abundance peptides/contaminants are present, competition for charge during the electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) processes can lead to ion suppression for the targeted analyte(s). In this note, a simple reversed-phase microcolumn sample separation and deposition device (Sep-Dep) is described. The use of this device improves or renders possible the analysis of complex or contaminated peptide mixtures by MALDI-MS. The method is simple and inexpensive and utilizes single-use low-cost Geloader-type columns packed with reversed-phase material. The device described utilizes an open column, allowing for a gradient or narrow-step gradient to be applied by any solvent delivery system or manually with a pipet. A key feature of the device is a deposition chamber that can be custom-built to hold any MALDI target. The Sep-Dep device is attached directly to an in-house vacuum line and draws solvent from the open-ended LC column. The elution of separated peptides is performed directly onto a target that has been treated with a hydrophobic barrier. This barrier effectively isolates fractions and improves the quality and morphology of the matrix crystals. The method produces efficient separations of proteolytic peptides, significantly reducing signal suppression effects in MALDI.

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column.

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C18 columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C18 RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.

Determination of gradient elution conditions for the separation of peptide mixtures by reversed-phase high-performance liquid chromatography: bovine β-casein tryptic digest

SummaryTaking as an example a tryptic hydrolysate of bovine β-casein, it was shown that the method used for the determination of optimal elution conditions for isocratic systems also applied to the separation of complex peptide mixtures by reversed-phase high-performance liquid chromatography with linear gradients. Using a C18 column and the same solvents, successive studies of the influence of flow rate, pH and temperature allowed a satisfactory separation of the sample in less than 30 min. Valuable information on the specificity of the action of trypsin on β-casein was deduced from the yield of the eluted peptides.

Separation of peptide mixtures by reversed-phase gradient elution. Use of flow rate changes for controlling band spacing and improving resolution

Separation of peptide mixtures by reversed-phase gradient elution. Use of flow rate changes for controlling band spacing and improving resolution

High-performance liquid chromatography in microsequencing

The tremendous success of protein structural determinations on a microscale would not have been possible without the introduction of high-performance liquid chromatography (HPLC) into the various steps of this procedure, i.e. separation of peptide mixtures, amino acid analysis and protein sequence analysis. In this review the advantages, limitations and current developments in the application of HPLC to protein structural analysis are discussed.

Fragmentation of proteins for sequence studies and separation of peptide mixtures.

When a protein has been judged homogeneous and the end groups and amino acid composition are known, the next step in the elucidation of the covalent structure is the degradation of the macromolecule into low molecular weight peptides which can be completely characterized with respect to composition and sequence. The entire composition of the protein should be accounted for by the summation of unique peptides. Once this is achieved, the sequenced peptides must be arranged in the same order as they occur in the intact protein. No single technique is ideally suited to accomplish this task; therefore, a combination of enzymic, or chemical and enzymic techniques, is required. The methods discussed in this chapter deal primarily with the fragmentation of proteins and the fractionation of the resulting peptide mixtures. With the exception of partial acid hydrolysis, the degradative procedures have been restricted to those which exhibit a reasonable degree of specificity and which are most often used to obtain sets of peptides necessary for the elucidation of the primary structure of a protein. Consequently, certain proteases, such as subtilisin, papain, pronase, and elastase which are extremely valuable for the hydrolysis of peptides, are discussed elsewhere in this book.