Identification of Peptide tumor markers in a tumor graft model in immunodeficient mice

The medical demand for useful biomarkers is large and still increasing. This is especially true for cancer, because for this disease adequate diagnostic markers with high specificity and sensitivity are still lacking. Despite advances in imaging technologies for early detection of cancer, peptidomic multiplex techniques evolved in recent years will provide new opportunities for detection of low molecular weight (LMW) proteome biomarker (peptides) by mass spectrometry. Improvements in peptidomics research were made based on separation of peptides and/or proteins by their physico-chemical properties in combination with mass spectrometric detection, respectively identification, and sophisticated bioinformatic tools for data analysis. To evaluate the potential of serological tumor marker detection by differential peptide display (DPD) we analyzed plasma samples from a tumor graft model. After subcutaneous injection of HCT-116 cells in immunodeficient mice and their growth to a palpable tumor, plasma samples were analyzed by DPD. The comparison of obtained mass spectrometric data allows discovery of tumor specific peptides which fit well into the biological context of cancer pathogenesis and show a strong correlation to tumor growth. The identified peptides indicate events associated with hyper-proliferation and dedifferentiation of cells from an epithelial origin, which are typical characteristics of human carcinomas. We conclude that these findings are a "proof of principle" to detect differentially expressed, tumor-related peptides in plasma of tumor-bearing mice.     

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

Photo-SRM: laser-induced dissociation improves detection selectivity of Selected Reaction Monitoring mode

Selected Reaction Monitoring (SRM) carried out on triple‐quadrupole mass spectrometers coupled to liquid chromatography has been a reference method to develop quantitative analysis of small molecules in biological or environmental matrices for years and is currently emerging as a promising tool in clinical proteomic. However, sensitive assays in complex matrices are often hampered by the presence of co‐eluted compounds that share redundant transitions with the target species. On‐the‐fly better selection of the precursor ion by high‐field asymmetric waveform ion mobility spectrometry (FAIMS) or increased quadrupole resolution is one way to escape from interferences. In the present work we document the potential interest of substituting classical gas‐collision activation mode by laser‐induced dissociation in the visible wavelength range to improve the specificity of the fragmentation step. Optimization of the laser beam pathway across the different quadrupoles to ensure high photo‐dissociation yield in Q2 without detectable fragmentation in Q1 was assessed with sucrose tagged with a push‐pull chromophore. Next, the proof of concept that photo‐SRM ensures more specific detection than does conventional collision‐induced dissociation (CID)‐based SRM was carried out with oxytocin peptide. Oxytocin was derivatized by the thiol‐reactive QSY® 7 C₅‐maleimide quencher on cysteine residues to shift its absorption property into the visible range. Photo‐SRM chromatograms of tagged oxytocin spiked in whole human plasma digest showed better detection specificity and sensitivity than CID, that resulted in extended calibration curve linearity. We anticipate that photo‐SRM might significantly improve the limit of quantification of classical SRM‐based assays targeting cysteine‐containing peptides. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of proteins copurifying with the cd4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies.     

Marker peptide selection for the determination of hazelnut by LC–MS/MS and occurrence in other nuts

The aim of this work was identifying and selecting hazelnut marker peptides and subsequently developing a complementary method of common immunoassay for the detection of hazelnut. For this purpose, at first, an in silico digestion of three major hazelnut allergens (Cor a 8, Cor a 9 and Cor a 11) was performed to get information about expected peptides. After extraction and trypsin digestion of hazelnut proteins, the samples were measured with tandem mass spectrometry (MS/MS) by direct infusion, which led to identification of 14 peptides. Eight of them with the highest MS signal were synthesized and used as standards for developing a liquid chromatography (LC)–MS/MS method in selected reaction monitoring (SRM) mode. Since almost all food allergens derived from nuts belong to the seed storage protein family and have homologue structure, a Basic Local Alignment Search Tool (BLAST) search was performed to identify the hazelnut specificity of the developed method. According to BLAST, only one peptide occurs in three other nuts, and the remaining seven selected peptides are hazelnut specific. Additionally to hazelnut, the eight other listed nuts in Directive 2003/89/EC as allergen were extracted, digested and measured with the developed method to prove the BLAST results. The analytical data confirmed that six peptides are hazelnut specific, on the contrary to anti-hazelnut antibodies, which showed cross-reactivities to all other nut extracts. Comparing these results, it could be shown that with this LC–MS/MS method in SRM mode, the specific detection of hazelnut is possible.     

Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.     

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.     

Comparison between a high‐resolution single‐stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs

The capabilities of a high‐resolution (HR), accurate mass spectrometer (Exactive‐MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50 000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive‐MS was compared with that of a triple quadrupole mass spectrometer (TQ‐MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole‐N‐oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR‐MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR‐MS offers several benefits over TQ‐MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR‐MS delivers comparable results to TQ‐MS in routine quantitative drug analyses. Considering the advantages of HR‐MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed. Copyright © 2012 John Wiley & Sons, Ltd.     

Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean-up

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.     

On-tissue protein identification and imaging by MALDI-Ion mobility mass spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.     

The use of proteotypic peptide libraries for protein identification

This paper describes an algorithm to apply proteotypic peptide sequence libraries to protein identifications performed using tandem mass spectrometry (MS/MS). Proteotypic peptides are those peptides in a protein sequence that are most likely to be confidently observed by current MS-based proteomics methods. Libraries of proteotypic peptide sequences were compiled from the Global Proteome Machine Database for Homo sapiens and Saccharomyces cerevisiae model species proteomes. These libraries were used to scan through collections of tandem mass spectra to discover which proteins were represented by the data sets, followed by detailed analysis of the spectra with the full protein sequences corresponding to the discovered proteotypic peptides. This algorithm (Proteotypic Peptide Profiling, or P3) resulted in sequence-to-spectrum matches comparable to those obtained by conventional protein identification algorithms using only full protein sequences, with a 20-fold reduction in the time required to perform the identification calculations. The proteotypic peptide libraries, the open source code for the implementation of the search algorithm and a website for using the software have been made freely available. Approximately 4% of the residues in the H. sapiens proteome were required in the proteotypic peptide library to successfully identify proteins.     

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.     

Optimization of microfabricated nanoliter-scale solid-phase extraction device for detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization mass spectrometry

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins.     

Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry

Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ_1–5, Aβ_6–16, Aβ_17–28 and Aβ_29–40(42) of either Aβ_1–40 or Aβ_1–42. Among those peptides, Aβ_17–28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL⁻¹ with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL⁻¹ of Aβ_1–40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.     

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL⁻¹. The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.     

Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis‐stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO‐mimetic peptides is under development from which peginesatide (also referred to as Hematide?), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase‐III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO‐mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL). Copyright © 2011 John Wiley & Sons, Ltd.     

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC)

Stable isotope labeling with amino acids in cell culture (SILAC) has evolved to be a major technique for quantitative proteomics using cell cultures. We developed a rapid method to follow and determine the incorporation of arginine and lysine. Analysis of the heavy state is required to avoid quantification errors. Moreover, the mixture of light and heavy states can be exploited to normalize the protein amount for subsequent relative quantification experiments. Therefore, peptides from different cell lines were extracted with 0.1% trifluoroacetic acid and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). This analysis was highly reproducible and was performed in less than 2 h, significantly faster than other methods for the same purpose. Similar peptide mass profiles were obtained for human EBV-transformed B, Jurkat T, and HeLa cells as well as for mouse embryonic fibroblasts. Proteolytic fragments of 27 human proteins were identified with 56 peptides by MALDI-MS/MS and can be used as a database for these kinds of experiments. Sequencing revealed that the peptides were predominantly amino- and carboxy-terminal protein fragments displaying a specificity characteristic of the acidic proteases cathepsin D and E. Many of the identified peptides contained arginine and/or lysine, allowing determination of the incorporation rate of these amino acids. Furthermore, the rate of conversion of arginine into proline could be monitored easily.