Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C(18)) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C(18)-SCX-C(18) stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C(18)- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C(18)-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C(18)-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.     

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.     

Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species

Summary A method was developed for plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation was achieved using 3% sodium alginate and 75 mM CaCl₂·2H₂O. Maximum percentage response for conversion of encapsulated shoot tips into plantlets was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant growth regulator. The regrowth ability of encapsulated shoot tips was affected by the concentration of sodium alginate, storage duration, and the presence or absence of MS nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were transferred to pots containing an autoclaved mixture of soilrite and peat moss (1∶1). The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts. Encapsulation of vegetative propagules in calcium alginate beads can be used as an alternative to synthetic seeds derived from somatic embryos.     

Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches

Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.     

Highly selective enrichment of phosphorylated peptides using titanium dioxide

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).     

Encapsulation-Dehydration and Encapsulation-Vitrification Based Cryopreservation of in vitro-Grown Shoot-Tips of Medicinal Plant Astragalus membranaceus

Shoot tips of Amembranaceus excised from in vitro grown axillary bud were encapsulated in calcium alginate beads. Subsequently, shoot tips were precultured in liquid MS medium enriched with 075mol·L-1 sucrose for 5d at 25℃ and then desiccated aseptically on dried silica gel for 5h to a water content of 231% (fresh weight basis) prior to immersion in liquid nitrogen (LN) for 1d. After rewarming at a 40℃ water bath for 2-3min and transferred to solid culture medium for shoot tip recovery. About 50% of cryopreserved shoot tips grew into shoots within 2 weeks after plating. Cryopreservation of Astragalus membranaceus (Fisch.) Bge. shoot tips by encapsulation vitrification has also been developed. Excised shoot tips were firstly encapsulated into alginate gel beads and then precultured in liquid MS medium containing 1mg·L-1 6 BA, 005mg·L-1 NAA and 075mol·L-1 sucrose at 25℃ for 3d. After loading for 90min with a mixture of 2mol·L-1 glycerol and 04mol·L-1 sucrose at 25℃, shoot tips were dehydrated with PVS2 for 120min at 0℃ prior to direct immersion in liquid nitrogen for 1d. After rapidly thawing at a 37℃ water bath for 2-3min, shoot tips were washed for 10min with liquid MS medium supplemented with 1mg·L-1 6 BA, 005mg·L-1 NAA and 12mol·L-1 sucrose at 25℃ and then post cultured on solid MS medium supplemented with 2mg·L-1 6 BA, 005mg·L-1 NAA. The regeneration rate of shoot tips amounted to nearly 80%. Both of plantlets regenerated from cryopreserved shoot tips were morphologically uniform, which both showed as that of control plants. Thus, this encapsulation dehydration and encapsulation vitrification technique appears promising as a routine method for the cryopreservation of shoot tips of Amembranaceus.     

Phosphatase-directed phosphorylation-site determination: a synthesis of methods for the detection and identification of phosphopeptides

Phosphopeptides can be difficult to detect and sequence by mass spectrometry (MS) due to low ionization efficiency and suppression effects in the MS mode, and insufficient fragmentation in the tandem MS (MS/MS) mode, respectively. To address this problem, we have developed a technique called Phosphatase-directed Phosphorylation-site Determination (PPD), which combines on-target phosphatase reactions, MALDI MS/MS of IMAC beads on target, and hypothesis-driven MS (HD-MS). In this method, on-target dephosphorylation experiments are conducted on IMAC-bound phosphopeptides, because dephosphorylated peptides have, in general, higher MS sensitivities than the corresponding phosphopeptides. The detected dephosphorylated peptides are sequenced by MS/MS, which identifies the potentially phosphorylated peptide and the total number of Ser, Thr, or Tyr residues that could hypothetically be phosphorylated within that peptide. On the basis of this information, a mass list containing every possible phosphorylation state of each observed peptide (where 1 HPO(3) = 80 Da) is used to direct MALDI-MS/MS on the phosphorylated peptides bound to IMAC beads at each theoretical mass from the list. If the peptide is present, the resulting MS/MS spectrum reveals the exact site(s) of phosphorylation in the peptide. We have demonstrated the applicability of PPD to the detection of in vivo phosphorylation sites on the Drosophila Stem Loop Binding Protein (dSLBP), and the complementarity of this new technique to conventional MS phosphorylation site mapping methods, since the phosphorylation sites in dSLBP could not be detected by other methods.     

Isotherm for Adsorption of Agrobacterium tumefaciens to Susceptible Potato (Solanum tuberosum L.) Tissues

Potato tuber disks were submerged in suspensions containing 10¹ to 10⁹ cells of Agrobacterium tumefaciens B6 per ml. After 60 min, the disks were rinsed and homogenized, and portions of the homogenates were plated to measure the number of adsorbed bacteria. At low initial bacterial concentrations (10⁵/ml), 5 to 23% of the bacteria adsorbed. At higher bacterial concentrations, the corresponding value was approximately 1.2%. Adsorption was a reversible equilibrium process. Binding saturation was not achieved, and adsorbed bacteria were confined to monolayers on the surfaces of tissue prepared for scanning electron microscopy. Adsorption of strain B6 to potato tuber tissues is described accurately by the Freundlich adsorption isotherm and may be a nonspecific phenomenon.     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant <Emphasis Type="Italic">Emmenopterys henryi</Emphasis> Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS₂) for 30 min at 0°C and followed by 100% PVS₂ for 40 min at 0°C. After changing the solution with fresh 100% PVS₂, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l⁻¹, α-naphthaleneacetic acid 0.1 mg l⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.     

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.     

New method for peptide desorption from abundant blood proteins for plasma/serum peptidome analyses by mass spectrometry

This report describes a new method for desorption of low-molecular weight (LMW) peptides from abundant blood proteins for use in subsequent mass spectrometry analyses. Heating of diluted blood serum to 98°C for 15min resulted in dissociation of LMW peptides from the most abundant blood proteins. Application of blood plasma/serum fractionation using magnetic beads with a functionalized surface followed by heating of the resultant fractions significantly increases the number of LMW peptides detected by MALDI-TOF MS, enhances the general reproducibility of mass spectrometry profiles and considerably increases the number of identified blood serum peptides by LC-MS/MS using an Agilent 6520 Accurate-Mass Q-TOF.     

Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles

Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.     

Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry

In vitro compartmentalisation in an emulsion was used to physically link proteins to the DNA that encodes them via microbeads. These microbeads can be selected for catalysis, or, as demonstrated here, for binding. Genes encoding a peptide containing an epitope (haemagglutinin) were enriched to near purity from a 10(6)-fold excess of genes encoding a different peptide by two rounds of selection using flow cytometry, indicating approximately 1000-fold enrichment per round. Single beads can be isolated using flow sorting and the single gene on the bead amplified by polymerase chain reaction. Hence, the entire process can be performed completely in vitro.     

A platform for high-throughput molecular characterization of recombinant monoclonal antibodies

We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF.     

Quantitative assays for esterified oxylipins generated by immune cells

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins--in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids--attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC-tandem mass spectrometry (LC/MS/MS) in multiple reaction-monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ～2 d. Following this, extraction of 30 samples takes ～3 h, with LC/MS/MS run time of 50 min per sample.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography

The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.     

Identification of all classes of spin-trapped carbon-centered radicals in soybean lipoxygenase-dependent lipid peroxidations of omega-6 polyunsaturated fatty acids via LC/ESR,LC/MS,and tandem MS

Using the combined techniques of on-line high performance liquid chromatography/electron spin resonance (LC/ESR) and mass spectrometry (MS), we previously identified spin-trapped adducts of all expected carbon-centered lipid-derived radicals ((*)L(d)) formed in linoleic acid peroxidation. In the present study, spin trapped lipid-derived carbon-centered radicals formed from the reactions of two omega-6 polyunsaturated fatty acids (PUFAs: linoleic and arachidonic acids) with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butyl nitrone (POBN) were identified using a combination of LC/ESR and LC/MS. All expected lipid-derived carbon-centered radicals in lipoxygenase-dependent peroxidations of linoleic acid and arachidonic acid were detected and identified by the combination of LC/ESR and LC/MS with confirmation by tandem mass spectrometry (MS/MS). The five classes of (*)L(d) formed from both omega-6 PUFAs including lipid alkyl radicals (L(*)), epoxyallyic radicals (OL(*)), dihydroxyallyic radicals ((*)L(OH)(2)), and a variety of R(*) and (*)RCOOH from beta-scission of lipid alkoxyl radicals, gave distinct retention times: POBN/(*)L(OH)(2) approximately 4-6 min, POBN/R(*) and POBN/(*)RCOOH approximately 8-22 min, POBN/L(*) and PBON/OL(*) approximately 25-36 min. The major beta-scission products in peroxidations of omega-6 PUFAs were the pentyl radicals. The ratio of beta-scission products, however, varied significantly depending on pH, [PUFA], as well as [O(2)].     

Online immunoaffinity LC/MS/MS. A general method to increase sensitivity and specificity: How do you do it and what do you need?

There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis. We describe here our methods and procedures for how to perform online IA-LC/MS/MS including a detailed protocol for the preparation of antibody (Ab) enrichment columns. We have included sample trapping and Ab methods. Furthermore, we highlight tips, tricks, minimal and optimal approaches. This technology has been shown to be viable for several applications, species and fluids from small molecules to proteins and biomarkers to PK assays.