Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry

Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of "fluorous proteomics," whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.     

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Metabolic profiling of flavonoids in Lotus japonicus using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry

Flavonoids detected from a model legume plant, Lotus japonicus accessions Miyakojima MG-20 and Gifu B-129, were profiled using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR/MS). Five flavonols and two anthocyanidins were detected as aglycones. LC-FTICR/MS facilitated simultaneous detection of 61 flavonoids including compounds that have not been reported previously. Chemical information of the peaks such as retention time, lambdamax, m/z value of the quasi-molecular ion, m/z value of MS/MS fragment ions, and relative intensity of MS/MS fragments was obtained, along with the molecular formulas and conjugate structures. Fourteen were completely identified by comparison with authentic compounds. The high accuracy of m/z values, being 0.081 ppm between observed and theoretical values, allowed prediction of molecular formulas of unknown compounds with the help of isotope peak information for determination of chemical composition. Based on a predicted elemental composition, the presence of a novel nitrogen-containing flavonoid was proposed. A comparison of flavonoid profiles in flowers, stems, and leaves demonstrated that the flowers yielded the most complex profile, containing 30 flower-specific flavonoids including gossypetin glycosides and isorhamnetin glycosides. A comparison of flavonoid profiles between MG-20 and B-129 grown under the same conditions revealed that the accumulation of anthocyanins was higher in B-129 than MG-20, particularly in the stem. Developmental changes in the flavonoid profiles demonstrated that kaempferol glycosides increased promptly after germination. In contrast, quercetin glycosides, predominant flavonoids in the seeds, were not detectable in growing leaves.     

Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability

To further our knowledge of intracellular copper transport, we used a proteomics strategy to search for hepatic proteins with copper-binding ability. Hep G2 cytosolic and microsomal fractions were applied to a copper(II)-loaded immobilized metal-affinity chromatography (IMAC) column. Protein identification was performed with 2-D gel electrophoresis and mass spectrometry. We identified 48 cytosolic proteins and 19 microsomal proteins displaying copper-binding ability. These proteins are diverse in function. Fifty-two of the 67 proteins contain putative metal-binding domains. We have identified many components of the Hep G2 copper metalloproteome including a large number of proteins not previously known to bind copper.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma

A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra? MS C₁₈ column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

New ways in qualitative and quantitative protein analysis: Nano chromatography coupled to element mass spectrometry

The potential of inductively coupled plasma-mass spectrometry (ICP-MS), which allows element-specific detection of heteroelements (e.g. Se and S) incorporated in protein structures, is highlighted for sensitive qualitative and quantitative protein analysis. ICP-MS coupled to separation techniques such as size exclusion chromatography and gel electrophoresis (via laser ablation) can be employed at different steps in the proteomic workflow. Special emphasis is made on the couplings of capillary and nanoHPLC to ICP-MS that required the development of dedicated interfaces. Element-specific peptide mapping by nanoHPLC–ICP-MS has turned out to be a key technique in combination with peptide sequencing via nanoHPLC–electrospray MS. This could impressively be demonstrated for the identification of selenium-containing proteins in selenium-rich yeast. Furthermore the potential of sulfur isotope dilution analysis in nanoHPLC–ICP-MS is presented as generic tool for highly accurate, absolute protein quantification.     

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002     

Determination of thioxylo-oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X-ray crystallography

Noncovalent binding of thioxylo-oligosaccharide inhibitors, methyl 4-thio-alpha-xylobioside (S-Xyl2-Me), methyl 4,4II-dithio-alpha-xylotrioside (S-Xyl3-Me), methyl 4,4II,4III-trithio-alpha-xylotetroside (S-Xyl4-Me), and methyl 4,4II,4III,4IV-tetrathio-alpha-xylopentoside (S-Xyl5-Me), to three family 11 endo-1,4-beta-xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS and X-ray crystallography. Ultra-high mass-resolving power and mass accuracy inherent to FT-ICR allowed mass measurements for noncovalent complexes to within |DeltaM|average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 10(4)-10(3) M-1, decreasing in the series of S-Xyl5-Me>or=S-Xyl4-Me>S-Xyl3-Me. In contrast, S-Xyl2-Me did not bind to any xylanase at the initial concentration of 5-200 microM, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three. The crystal structures of CTX-inhibitor complexes gave interesting insights into the binding. Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site. The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex with beta-d-xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci3, 465-475, and Sabini E, Wilson KS, Danielsen S, Schulein M & Davies GJ (2001) Acta CrystallogrD57, 1344-1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.     

Improved identification of monomethyl paraffin chain branching (close to the methyl end) of long-chain compounds by gas chromatography and mass spectrometry

An improved method for the identification of monomethyl substituted paraffin chains (n?2, n?3 and n?4) by gas chromatography and mass spectrometry is presented. Fatty acids and sphingolipid long-chain bases were converted to their corresponding alcohols, which were analyzed as methyl ethers. These compounds were better separated on packed columns (XE-60) than other derivatives, and the low temperature of analysis minimized the bleeding of stationary phase into the mass spectrometer. Mass spectra of methyl ethers allowed a more conclusive identification of branches than of other derivatives. The method may be generally applied to fatty acids, aldehydes and alcohols.     

Isocratic solid phase extraction-liquid chromatography (SPE-LC) interfaced to high-performance tandem mass spectrometry for rapid protein identification

Reversed-phase liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) allows analysis of very complex peptide mixtures at great sensitivity, but it can be very time-consuming, typically using 60 min, or more, per sample analysis. We recently introduced the isocratic solid phase extraction-liquid chromatography (SPE-LC) technology for rapid separation (~8 min) of simple peptide samples. We now extend these studies to demonstrate the potential of SPE-LC separation in combination with a hybrid linear ion trap-Orbitrap tandem mass spectrometer for efficient analysis of peptide samples in proteomics research. The system performance of SPE-LC-MS/MS was evaluated in terms of sensitivity and efficiency for the analysis of tryptic peptide digests obtained from samples consisting of up to 12 standard proteins. The practical utility of the analytical setup was demonstrated by the analysis of <15 microg depleted human serum proteome by a combination of SDS-PAGE and SPE-LC-MS/MS. A total of 88 unique gene products spanning 3 orders of magnitude in serum protein concentration were identified using stringent database search criteria.     

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25 pM with the linearity of the standard curve spanned to 2500 pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.     

Respective contributions of atomic emission spectrometry (AES) and secondary ion mass spectrometry (SIMS) to trace element quantification

By means of Secondary Ion Mass Spectrometry (SIMS) it is possible to measure in situ the relative concentration of a given element in a volume of 1 micron 3. Atomic Emission Spectrometry (AES) allows absolute quantitation of tissue homogenates. The use of both techniques lead to correlate relative and absolute elemental concentrations. These methods have been applied to lithium and manganese quantitation after treatments at a therapeutic dose. The results assess the sensibility of SIMS analysis, around 0.1 ppm in biological specimens, and confirm the adequacy of the instrument to trace elements study.     

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or "sphingolipidomic" methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.     

Markers of protein oxidation: different oxidants give rise to variable yields of bound and released carbonyl products

Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed on the C-3 carbons of Ala, Val, Leu, and Asp residues undergo beta-scission to give backbone alpha-carbon radicals, with the release of the side- chain as a carbonyl compound. We now show that this is a general mechanism that occurs with a wide range of oxidants. The quantitative significance of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride, and a peroxynitrite generator, 3-morpholinosydnonimine hydrochloride, gave lower overall carbonyl yields, with released carbonyls predominating over protein-bound species similar to that observed with metal ion/H2O2 systems.     

PROXIMO--a new docking algorithm to model protein complexes using data from radical probe mass spectrometry (RP-MS)

The design and implementation of a new algorithm, known as PROXIMO for protein oxidation interface modeller, is described to predict the structure of protein complexes using data generated in radical probe mass spectrometry (RP-MS) experiments. Photochemical radiolysis and discharge sources can be used to effect RP-MS in which hydroxyl radicals are formed directly from the bulk solvent on millisecond timescales and react with surface accessible residues in footprinting-like experiments. The algorithm utilizes a geometric surface fitting routine to predict likely structures for protein complexes. These structures are scored based on a correlation between the measured solvent accessibility of oxidizable residue side chains and oxidation shielding data obtained by RP-MS. The algorithm has been implemented to predict structures for the ribonuclease S-protein-peptide and calmodulin-melittin complexes using RP-MS data generated in this laboratory. The former is in close agreement with the high-resolution experimental structure available.     

Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS)

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.     

Preparation and mass spectrometry of 14 pure and 18O(2)-labeled oxidation products from the phytosterols beta-sitosterol and stigmasterol

To lower cholesterol, phytosterols are currently introduced as food additives, where they may become oxidized. In addition, specific biological effects of oxyphytosterols are suggested by the recent molecular clarification of the phytosterol storage disease as a dysfunctional mutation of an active sterol reexporter potentially regulated by oxidized phytosterols. We therefore studied the hydroxybenzotriazole-mediated PbO(2)-driven oxidation of phytosterols and compared it to the oxidation of cholesterol. We prepared, identified, and purified standards of 14 oxidation products of two major phytosterols. The gas chromatographic mass spectrometric characteristics of the 7alpha- and 7beta-hydroxy-, 5alpha,6alpha-epoxy, 5beta,6beta-epoxy, 7keto-, 3beta,5alpha,6beta-trihydroxy-, 3keto-, and 7-dehydro-derivatives of beta-sitosterol and stigmasterol are presented. The method also provided a convenient access to prepare 18O-labeled oxyphytosterols of high chemical and isotopic purity and can easily be extended to further phytosterols and -stanols. This enables the gas chromatography-mass spectrometry analysis of oxyphytosterols and the study of their biological effects.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate dimethyl benzoylphenylurea (BPU) and its five metabolites in human plasma and urine for clinical pharmacology studies

A method has been developed for the quantitation of N-[4-(5-bromo-2-pyrimidinyloxy)-3-methylphenyl]-N'-(2-dimethylamino-benzoyl)urea (BPU) and its metabolites in human plasma and urine. BPU and metabolites were separated on a C18 column with acetonitrile-water mobile phase containing 0.1% formic acid using isocratic flow for 5 min. The analytes were monitored by tandem mass spectrometry. Calibration curves were generated over the range of 2.5-500 ng/mL for BPU, mmBPU, and aminoBPU in plasma; and 0.1-20, 0.1-20, 0.5-100, 10-2000, 1-200, and 3-600 ng/mL for BPU, mmBPU, aminoBPU, G280, G308, and G322 in urine, respectively. The method has been successfully applied to study the pharmacokinetics of BPU.