Peptide Elution Research Articles

A Novel Prefractionation Method Combining Protein and Peptide Isoelectric Focusing in Immobilized pH Gradient Strips

To increase sensitivity and analytical depth in shotgun proteomics, prefractionation of complex samples is often used. Here we describe a novel prefractionation method, Sandwich high resolution isoelectric focusing, which combines both protein and peptide isoelectric focusing. In the first step, intact proteins are separated on the basis of isoelectric point (pI) using traditional immobilized pH gradient (IPG) strips. Segments in the IPG-strip containing proteins of interest are subsequently cut out and applied to in-strip digestion, without subsequent peptide elution. In the second peptide isoelectric focusing step, the strip segments are used as loading bridges. The peptides are thereby directly applied to the peptide isoelectric focusing, without an intermediate elution step, and separated on narrow range IPG strips to reduce the complexity on the peptide level. In the final step, the peptides are eluted into 96-well plates and analyzed with mass spectrometry. In a proof of principle experiment, using this method to zoom in on pI regions of interest in human plasma, we identify over 800 proteins, with concentrations spanning over 6 orders of magnitude.

Inversion of chromatographic elution orders of peptides and its importance for proteomics

Inversion of the order of peptide elution in reversed-phase liquid chromatography under changing separation conditions, such as gradient slope has been considered. Using a six-protein proteolytic peptide standard and available literature data, the occurrence frequency and importance of this phenomenon in proteomic studies utilizing methods of shotgun proteomics and accurate mass and time tags have been evaluated. Feasibility of qualitative and quantitative description of peptide elution order inversion has been demonstrated using a model of critical liquid chromatography. Existing approaches to predict peptide separation and directions of the shifts of chromatographic peaks when the gradient profile changes have been compared.

SIMPATIQCO: A Server-Based Software Suite Which Facilitates Monitoring the Time Course of LC–MS Performance Metrics on Orbitrap Instruments

While the performance of liquid chromatography (LC) and mass spectrometry (MS) instrumentation continues to increase, applications such as analyses of complete or near-complete proteomes and quantitative studies require constant and optimal system performance. For this reason, research laboratories and core facilities alike are recommended to implement quality control (QC) measures as part of their routine workflows. Many laboratories perform sporadic quality control checks. However, successive and systematic longitudinal monitoring of system performance would be facilitated by dedicated automatic or semiautomatic software solutions that aid an effortless analysis and display of QC metrics over time. We present the software package SIMPATIQCO (SIMPle AuTomatIc Quality COntrol) designed for evaluation of data from LTQ Orbitrap, Q-Exactive, LTQ FT, and LTQ instruments. A centralized SIMPATIQCO server can process QC data from multiple instruments. The software calculates QC metrics supervising every step of data acquisition from LC and electrospray to MS. For each QC metric the software learns the range indicating adequate system performance from the uploaded data using robust statistics. Results are stored in a database and can be displayed in a comfortable manner from any computer in the laboratory via a web browser. QC data can be monitored for individual LC runs as well as plotted over time. SIMPATIQCO thus assists the longitudinal monitoring of important QC metrics such as peptide elution times, peak widths, intensities, total ion current (TIC) as well as sensitivity, and overall LC–MS system performance; in this way the software also helps identify potential problems. The SIMPATIQCO software package is available free of charge.

Comparison of Orthogonality Estimation Methods for the Two-Dimensional Separations of Peptides

In two-dimensional chromatography, the orthogonality of separation is important for achieving high peak capacity. In this paper, a number of different metrics are compared as measures of orthogonality. Six peptide elution data sets acquired on different stationary phases are plotted against reversed phase retention data and examined as two-dimensional chromatographic pairs. The data, including six in silico prepared data pairs, are utilized to challenge and compare selected orthogonality metrics. The metrics include correlation coefficients, mutual information, box-counting dimensionality, and surface fractional coverage with different hulls. Although correlation coefficients were found to be less suited for the intended purpose, other methods can provide a suitable measure of orthogonality. The presented results are discussed in terms of method utility, simplicity, and applicability for statistically small sets of chromatographic data. Two of the methods, box counting dimensionality and fractional coverage, were found to be mathematically related.

Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells

An acoustophoresis-based microfluidic flow-chip is presented as a novel platform to facilitate analysis of proteins and peptides loosely bound to the surface of beads or cells. The chip allows for direct removal of the background surrounding the beads or cells, followed by sequential treatment and collection of a sequence of up to five different buffer conditions. During this treatment, the beads/cells are retained in a single flow by acoustic radiation force. Eluted peptides are collected from the outlets and subsequently purified by miniaturized solid-phase extraction and analyzed with matrix assisted laser desorption mass spectrometry. Fundamental parameters such as the system fluidics and dispersion are presented. The device was successfully applied for wash and sequential elution of peptides bound to the surface of microbeads and human spermatozoa, respectively.

A sensitive gel-based global O-glycomics approach reveals high levels of mannosyl glycans in the high mass region of the mouse brain proteome

We developed a gel-based global O-glycomics method applicable for highly complex protein mixtures entrapped in discontinuous gradient gel layers. The protocol is based on in-gel proteolysis with pronase followed by (glyco)peptide elution and off-gel reductive β-elimination. The protocol offers robust performance with sensitivity in the low picomolar range, is compatible with gel-based proteomics, and shows superior performance in global applications in comparison with workflows eliminating glycans in-gel or from electroblotted glycoproteins. By applying this method, we analyzed the O-glycome of human myoblasts and of the mouse brain O-glycoproteome. After semipreparative separation of mouse brain proteins by one-dimensional SDS gel electrophoresis, the O-glycans from proteins in different mass ranges were characterized with a focus on O-mannose-based glycans. The relative proportion of the latter, which generally represent a rare modification, increases to comparatively high levels in the mouse brain proteome in dependence of increasing protein masses.

Proteome Sampling by the HLA Class I Antigen Processing Pathway

The peptide repertoire that is presented by the set of HLA class I molecules of an individual is formed by the different players of the antigen processing pathway and the stringent binding environment of the HLA class I molecules. Peptide elution studies have shown that only a subset of the human proteome is sampled by the antigen processing machinery and represented on the cell surface. In our study, we quantified the role of each factor relevant in shaping the HLA class I peptide repertoire by combining peptide elution data, in silico predictions of antigen processing and presentation, and data on gene expression and protein abundance. Our results indicate that gene expression level, protein abundance, and rate of potential binding peptides per protein have a clear impact on sampling probability. Furthermore, once a protein is available for the antigen processing machinery in sufficient amounts, C-terminal processing efficiency and binding affinity to the HLA class I molecule determine the identity of the presented peptides. Having studied the impact of each of these factors separately, we subsequently combined all factors in a logistic regression model in order to quantify their relative impact. This model demonstrated the superiority of protein abundance over gene expression level in predicting sampling probability. Being able to discriminate between sampled and non-sampled proteins to a significant degree, our approach can potentially be used to predict the sampling probability of self proteins and of pathogen-derived proteins, which is of importance for the identification of autoimmune antigens and vaccination targets.

Instant spectral assignment for advanced decision tree-driven mass spectrometry

We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ∼16ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) real-time prediction of peptide elution windows en masse (∼3min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β‐amyloid peptide revealed by affinity mass spectrometry and molecular modeling

Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.

Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics

In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy. For amino labeling, cysteinyl peptides were immobilized on Sepharose-6B resin and labeled with DOTA-NHS ester followed by metallation with lanthanides. Thiol labeling was carried out using lanthanide-containing metal-coded affinity tags (MeCAT) after elution of peptides from the resin. Complete dual labeling of the standard peptides was demonstrated by liquid chromatography electrospray ionization mass spectrometry, whereas more than 80% of the detected peptides of BSA and HSA were completely dual-labeled. Parallel detection by LC coupled to inductively coupled plasma mass spectrometry (ICP-MS) delivered reliable quantitative information. Thus, the versatile lanthanide choice in both labeling steps allowed estimating primary amino and thiol stoichiometries for the studied samples using different lanthanides. On the other hand, enhancement of ICP-MS signal was achieved as expected when all positions were labeled with the same lanthanide. Finally, linear calibrations of the signal for most of the labeled peptides by standard additions of digested BSA showed a suitable behaviour for quantitative applications and demonstrated the pre-concentration capability of the employed resin.

Consecutive Proteolytic Digestion in an Enzyme Reactor Increases Depth of Proteomic and Phosphoproteomic Analysis

Analytical advantages of using multiple enzymes for sample digestion (MED), primarily an increase of sequence coverage, have been reported in several studies. However, this approach is only rarely used, mainly because it requires additional sample and mass spectrometric measurement time. We have previously described Filter Aided Sample Preparation (FASP), a type of proteomic reactor, in which samples dissolved in sodium dodecyl sulfate (SDS) are digested in an ultrafiltration unit. In FASP, such as in any other preparation protocol, a portion of sample remains after digestion and peptide elution. Making use of this fact, we here develop a protocol enabling consecutive digestion of the sample with two or three enzymes. By use of the FASP method, peptides are liberated after each digestion step and remaining material is subsequently cleaved with the next proteinase. We observed excellent performance of the ultrafiltration devices in this mode, allowing efficient separation of orthogonal populations of peptides, resulting in an increase in the numbers of identified peptides and proteins. At the low microgram level, we found that the consecutive use of endoproteinases LysC and trypsin enabled identification of up to 40% more proteins and phosphorylation sites in comparison to the commonly used one-step tryptic digestion. MED-FASP offers efficient exploration of previously unused sample material, increasing depth of proteomic analyses and sequence coverage.

Rapid Identification of Low Level Glycation Sites in Recombinant Antibodies by Isotopic Labeling with 13C6-Reducing Sugars

Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of (12)C(6)/(13)C(6) reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent. This approach simplified the detection of glycated tryptic peptide elution in the LC/MS analysis by giving a unique signature of two molecular ions with equal intensity and differing by 6.018 Da. An in-house developed script automatically processed large data files to generate a list of such peptide mass pairs. The high mass accuracy of the Orbitrap allowed us to assign the sequences unambiguously by comparison with all possible glycated peptide masses. This sequence list was subsequently used to verify their presence/absence in the digest of the native antibody. This work flow enabled rapid and confident identification of site-specific glycation even when levels are below 0.5%. We found the glycation sites to be distributed across the entire antibody studied.

Displacement chromatography as first separating step in online two-dimensional liquid chromatography coupled to mass spectrometry analysis of a complex protein sample—The proteome of neutrophils

Displacement chromatography provides some advantages over elution chromatography such as the opportunity to enrich trace amounts of molecules and to elute molecules in highest concentrations achievable with liquid chromatography. In a previous study we demonstrated that displacement chromatography is a well-suited alternative to gradient elution in an offline two-dimensional (2D-)LC–MS approach for the analysis of proteomes. In this study we present a method for applying displacement chromatography in an online 2D-LC–MS system including a cation exchange (CEX) column and a reversed phase column. We circumvented the problem of determining the sample capacity of the CEX column by repeated injection (pulses) of sample aliquots monitored by an LC–MS analysis of each flow-through fraction of the CEX column. Elution of tryptic peptides from the CEX column was achieved by repeated injection (pulses) of the displacer spermine. Pulsed displacer injections offer the advantage through physical separation of preventing post-column mixing of already separated compounds. As a proof of principle we analyzed the cytosolic proteome of human neutrophils.

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) versus Strong Cation Exchange (SCX) for Fractionation of iTRAQ-Labeled Peptides

The iTRAQ technique is popular for the comparative analysis of proteins in different complex samples. To increase the dynamic range and sensitivity of peptide identification in shotgun proteomics, SCX chromatography is generally used for the fractionation of iTRAQ-labeled peptides before LC-MS/MS analysis. However, SCX suffers from clustering of similarly charged peptides and the need to desalt fractions. In this report, SCX is compared with the alternative ERLIC method for fractionating iTRAQ-labeled peptides. The simultaneous effect of electrostatic repulsion and hydrophilic interaction in ERLIC results in peptide elution in order of decreasing pI and GRAVY values (increasing polarity). Volatile solvents can be used. We applied ERLIC to iTRAQ-labeled peptides from rat liver tissue, and 2745 proteins and 30,016 unique peptides were identified with high confidence from three technical replicates. This was 12.9 and 49.4% higher, respectively, than was obtained using SCX. In addition, ERLIC is appreciably better at the identification of highly hydrophobic peptides. The results indicate that ERLIC is a more convenient and more effective alternative to SCX for the fractionation of iTRAQ-labeled peptides. Quantification data show that both SCX and ERLIC fractionation have no significant effect on protein quantification by iTRAQ.

Processing methods for signal suppression of FTMS data

BackgroundFourier Transform Mass Spectrometry coupled with Liquid Chromatography(LC-FTMS) has been widely used in proteomics. Past investigation has revealed that there exists an intensity dependent random suppression in peptide elution profiles in LC-FTMS data. The suppression is homogenous for the same peptide but non-homogenous for different peptides. The correction of suppressed profiles and an estimation on the range of suppression are necessary for accurate and reliable quantification using FTMS data.ResultsA software package, Gcorr, is presented. The software corrects peptide profiles that satisfy correction conditions, and it can predict fold change null distributions at different intensity levels. Subsequently, the significance P-values of measured fold changes can be estimated based on the predicted null distributions. We have used an 1:1 LC-FTMS label-free dataset pair collected based on the same sample to verify that our predicted null distributions conforms to that of the observed null distribution.ConclusionsThis software is able to provide suppression correction for peptide profiles, suppression distribution analysis and peptide differential expression analysis in terms of its fold change significance. The software is freely available at http://compgenomics.utsa.edu/Suppression_Study.html.

Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04–0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.

Kinetics of Angiotensin I alteration of conformation on different hydrophobic interaction chromatographic surfaces

In the present study, Angiotensin I (Ang I) will be used as model peptide to assess on-column alteration of conformation phenomena. Adsorptive behavior of Ang I on various commercial hydrophobic interaction surfaces (Butyl, Octyl and Phenyl – Sepharose), under different conditions, was investigated. In order to calculate the cis–trans isomerization rate constants of Ang I on the stationary phase's surface, the first and second moments of the proline peptide elution profiles were determined. The activation energies for the isomerization process on Phenyl and Butyl Sepharose were also calculated. Results suggest that the stationary phase catalyzes Ang I isomerization and that catalysis is dependent on hydrophobic interaction ligand nature.

HLA class I allele promiscuity revisited

The peptide repertoire presented on human leukocyte antigen (HLA) class I molecules is largely determined by the structure of the peptide binding groove. It is expected that the molecules having similar grooves (i.e., belonging to the same supertype) might present similar/overlapping peptides. However, the extent of promiscuity among HLA class I ligands remains controversial: while in many studies T cell responses are detected against epitopes presented by alternative molecules across HLA class I supertypes and loci, peptide elution studies report minute overlaps between the peptide repertoires of even related HLA molecules. To get more insight into the promiscuous peptide binding by HLA molecules, we analyzed the HLA peptide binding data from the large epitope repository, Immune Epitope Database (IEDB), and further performed in silico analysis to estimate the promiscuity at the population level. Both analyses suggest that an unexpectedly large fraction of HLA ligands (>50%) bind two or more HLA molecules, often across supertype or even loci. These results suggest that different HLA class I molecules can nevertheless present largely overlapping peptide sets, and that “functional” HLA polymorphism on individual and population level is probably much lower than previously anticipated.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0552-6) contains supplementary material, which is available to authorized users.

Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.