EJMS protocol: systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometry-based phosphopeptide stability estimations?

There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.     

Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.     

Micropipette tips--the unsung heroes of mass spectrometry

Micropipette tip (tip) pipetting accuracy and precision are functions of tip manufacture consistency, morphological variation, and retained inner tip wall particulate matter. Irregular tip inner wall surfaces and retained particulate matter cause pipetting inaccuracy. Washed and unwashed tips from seven manufacturers were compared using weight by difference (six), microscopic Luxol Fast Blue staining (seven), and matrix-assisted laser desorption/onization time-of-flight (MALDI-TOF) mass spectrometry (six). Photomicrographs revealed tip irregularity and inner wall retained particulate matter. Computer model analysis was used to identify tip irregularity and retained matter. These tests established the utility of a pipette solvent wash to increase the performance and the accuracy of tips and, thus, improve the MALDI mass spectra obtained.     

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO₂‐MOAC) is widely regarded as being more selective than immobilized metal‐ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO₂‐MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO₂‐MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non‐phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5‐dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO₂‐MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO₂‐MOAC showed higher specificity than immobilized gallium (Ga³⁺), immobilized iron (Fe³⁺), or zirconium dioxide (ZrO₂) affinity chromatography for phosphopeptide enrichment. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.     

天冬氨酸作为非特异性吸附抑制剂在二氧化钛选择性富集磷酸肽中的应用

二氧化钛富集法作为目前使用最为广泛的金属氧化物富集磷酸肽的方法,在富集过程中常常对富含天冬氨酸和谷氨酸的酸性非磷酸化肽段存在一定的非特异性吸附作用。这些肽段与磷酸化肽段一同被富集,降低了磷酸肽富集的选择性。传统方法中使用的非特异性吸附抑制剂常会对质谱的电喷雾离子源造成污染,因而限制了其在液相色谱-质谱联用(LC-MS)系统中的应用。本研究将天冬氨酸作为一种新型的非特异性吸附抑制剂加入到二氧化钛富集体系中,并分别对3种和9种标准蛋白质酶切肽段混合物进行富集实验,同时与添加另一种非特异性吸附抑制剂——谷氨酸以及不添加任何非特异性吸附抑制剂的富集体系进行了富集效果的比较。结果表明,天冬氨酸可以有效地提高二氧化钛对磷酸肽富集的选择性。将添加天冬氨酸的二氧化钛富集体系应用于鼠肝全蛋白质磷酸肽的富集中,同样取得了很好的效果,表明天冬氨酸在复杂的生物样本的磷酸肽富集中也同样具有良好的应用前景。此外,由于天冬氨酸在反相色谱中极易被洗脱去除,从而避免了传统抑制剂对LC-MS系统离子源的污染问题。     

Efficient enrichment and desalting of protein digests using magnetic mesocellular carbon foams in matrix-assisted laser desorption/ionization mass spectrometry

We demonstrate that magnetic mesocellular carbon foams (Mag-MCF-C) can be effectively used for enrichment and desalting of protein digests or peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The large mesocellular pores and surface area of Mag-MCF-C are likely to mainly contribute to high efficiency in enrichment and desalting of protein digests. The magnetic property of Mag-MCF-C enabled easy and simple enrichment and desalting process comprising adsorption, washing, and separation steps by using an external magnet. Following elution from Mag-MCF-C by using a matrix solution (CHCA in 70% ACN/0.1% TFA), the peptides were subjected to MALDI-MS analysis. As a result, MALDI mass spectra of peptides or tryptic protein digests were distinct even at a peptide concentration as low as 50 pM. The use of Mag-MCF-C resulted in significantly improved sequence coverage for protein identification when compared to other conventional methods. Mag-MCF-C will find applications in mass spectrometric analysis of low abundance peptides or protein digests with high sensitivity.     

基于离心式富集装置的酪氨酸磷酸肽分析新方法

酪氨酸磷酸化及其相应激酶活性的研究在抗肿瘤药物靶点的研发中具有重要意义.由于酪氨酸磷酸化仅占蛋白质总磷酸化含量的不足0.1%,因此规模化的酪氨酸磷酸化鉴定面临着重大技术挑战.本研究构建了TiO2串联C18反相填料的离心式富集装置,结合抗体免疫沉淀法,建立了酪氨酸磷酸肽的富集策略.此新型富集装置由吸头、适配器和离心(EP)管组成,将TiO2富集磷酸肽和C18填料反相分离磷酸肽有机结合,以离心的方式进行样品的上样、清洗、洗脱和分离,再通过抗酪氨酸磷酸化抗体进一步特异性富集酪氨酸磷酸肽,从而实现了酪氨酸磷酸肽的高效富集和大规模质谱鉴定.通过离心式富集装置简化了实验步骤,减少了样品损失和人为因素干扰;而且离心式、平行化的样品处理方式可显著提高分析通量.将此策略成功用于小鼠肝脏蛋白质酪氨酸磷酸化肽段的富集和质谱鉴定,在5 mg鼠肝蛋白中共鉴定出967个酪氨酸磷酸化位点,对应545个蛋白质,显示了其在蛋白质组学研究中的应用潜力.     

Coupling of TiO(2)-mediated enrichment and on-bead guanidinoethanethiol labeling for effective phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry

Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO2 beads without the use of any hydroxy acid additives like 2,5-dihydroxybenzoic acid. Labeling was accomplished on-bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET-labeled derivatives were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine-mimic. In particular, GET-labeled lysine-terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on-bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures.     

Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC–ESI-MS/MS.     

Characterisation and evaluation of metal-loaded iminodiacetic acid-silica of different porosity for the selective enrichment of phosphopeptides

Silica particles of different porosity were functionalised with iminodiacetic acid (IDA) and loaded with Fe(III) to yield immobilised metal affinity chromatography stationary phases (Fe(III)-IDA-silica) for phosphopeptide enrichment. The elution step of bound phosphopeptides was optimised with a 32P radioactive labelled peptide by a comprehensive study. Several elution systems, including phosphate buffers of different pH and concentration and ethylenediaminetetraacetic acid solutions were employed. Furthermore the effect of support porosity on elution behaviour was investigated. Under best conditions recoveries higher than 90% were achieved. A solid-phase extraction (SPE) protocol was developed for fractionation of phosphorylated and non-phosphorylated peptides and desalting of the fractions which is essential for subsequent mass spectrometric analysis by the combination of Fe(III)-IDA-silica and C18-silica particles. The pH of the loading buffer was found to be a critical parameter for the efficiency of the SPE protocol. As tryptic digests of alpha-lactalbumin, lysozyme and ribonuclease A mixed with three synthetic phosphopeptides were fractionated, pH 2.5 provided minimal proportion of unspecific bound peptides when comparing the fractions after mu-LC-electrospray ionization MS separation. The effect of a sample derivatisation reaction (methylation) on the efficiency of phosphopeptide enrichment was further investigated. Blocking carboxylate groups by methyl ester formation totally prevented unspecific interaction with the immobilised Fe(III) ions, but generated partially methylated phosphopeptides that increased the complexity of the phosphorylated fraction.     

On particle ionization/enrichment of multifunctional nanoprobes: washing/separation-free, acceleration and enrichment of microwave-assisted tryptic digestion of proteins via bare TiO2 nanoparticles in ESI-MS and comparing to MALDI-MS

A simple, rapid, straightforward and washing/separation free of in-solution digestion method for microwave-assisted tryptic digestion of proteins (cytochrome c, lysozyme and myoglobin) using bare TiO(2) nanoparticles (NPs) prepared in aqueous solution to serve as multifunctional nanoprobes in electrospray ionization mass spectrometry (ESI-MS) was demonstrated. The current approach is termed as 'on particle ionization/enrichment (OPIE)' and it can be applied in ESI-MS, atmospheric pressure-matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The bare TiO(2) NPs can assist, accelerate and effectively enhance the digestion efficiency, sequence coverage and detection sensitivity of peptides for the microwave-assisted tryptic digestion of proteins in ESI-MS. The reason is attributed to the fact that proteins or partially digested proteins are easily attracted or concentrated onto the surface of TiO(2) NPs, resulting in higher efficiency of digestion reactions in the microwave experiments. Besides, the TiO(2) NPs could act as a microwave absorber to accelerate and enrich the protein fragments in a short period of time (40-60 s) from the microwave experiments in ESI-MS. Furthermore, the bare TiO(2) NPs prepared in aqueous solution exhibit high adsorption capability toward the protein fragments (peptides); thus, the OPIE approach for detecting the digested protein fragments via ESI and MALDI ionization could be achieved. The current technique is also a washing and separation-free technique for accelerating and enriching microwave-assisted tryptic digestion of proteins in the ESI-MS and MALDI-MS. It exhibits potential to be widely applied to biotechnology and proteome research in the near future.     

Photopolymerized microtips for sample preparation in proteomic analysis

We demonstrate a novel method for the fabrication of disposable plastic microtips, which we name "EasyTip", by a photopolymerization technique. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The fabricated EasyTips can be manipulated using commercial pipettes for wash/elution of minute amount of biological samples (< 10 microL) and can be applied for mass spectrometry (MS)-based proteomic analysis, in which the detection sensitivity depends critically on the optimal sample preparation. The recovery of a sample of 25 fmol of tryptic hemoglobin digest loaded in a C18 EasyTip was near 100% and we estimated the loading capacity to be around 0.4-2.0 microg of total proteins or peptides, which is well above a sufficient quantity for MS analysis. The effectiveness of the C18 EasyTips in enhancing the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MS signal, and thus providing a greater sequence coverage, was also demonstrated by the analysis of hemoglobin digest and the in-gel digested epidermal growth factor receptor (EGFR) protein from A431 cell lysate. We also demonstrated the usefulness of the immobilized IMAC EasyTips in extracting the signal of tryptic phosphopeptides of beta-casein (10 pmol) having one and four phosphorylation sites by using an IMAC EasyTip prior to off-line analysis by MS. The combination of IMAC EasyTips and MALDI-MS allowed the unambiguous identification of phosphopeptides based on the phosphatase assay as well as the post-source decay. Compared to other miniaturized devices, this fabrication method is simple, cheap, and requires less human intervention. Moreover, the method of manipulating the EasyTips is straightforward and can be automated readily by a robotic system for high-throughput analysis.     

Development of core–shell structure Fe3O4@Ta2O5 microspheres for selective enrichment of phosphopeptides for mass spectrometry analysis

Protein phosphorylation is one of the most important post-translational modifications. Due to the dynamic nature and low stoichiometry of the protein phosphorylation, enrichment of phosphopeptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Many metal oxides such as titanium dioxide and zirconium dioxide have been successfully applied to isolation and enrichment of phosphopeptides. Recently, niobium pentoxide was proved to have the ability for selective enrichment of phosphopeptides. Considering the proximity of tantalum to niobium, we supposed that Ta₂O₅ can be used as affinity probes for phosphopeptide enrichment. In the work, we synthesized Fe₃O₄@Ta₂O₅ magnetic microspheres with core–shell structure for selective enrichment of phosphopeptides. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe₃O₄@Ta₂O₅ magnetic microspheres to isolation and enrichment of the phosphopeptides from tryptic digestion of standard proteins and real samples, and then the enriched peptides were analyzed by matrix-assisted laser desorption mass spectrometry analysis (MALDI-MS) or liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS). Experiment results demonstrate that Ta₂O₅ coated-magnetic microspheres show the excellent potential for selective enrichment of phosphopeptides.     

Fast trypsin digestion of proteins on a cross‐linked [Os(dmebpy)2Cl]+/2+‐derivatized copolymer of acrylamide and vinylimidazole column

Fast digestion of proteins was observed when they were loaded together with trypsin onto the cross‐linked [Os(dmebpy)₂Cl]^+/2+‐derivatized copolymer of acrylamide and vinylimidazole column. The insoluble Os‐complexed polymer particles were packed into an electrospray tip to monitor peptides eluted during loading, washing and elution periods with a mass spectrometer. The proteolytic cleavage of proteins was observed immediately when the mixture of trypsin and substrates in 0.2 mM ammonium bicarbonate 50:50 H₂O/acetonitrile reached the column tip, and continued through the loading period. Some tryptic peptides were released from the column during the loading and following washing periods. The others still stayed on the column until the low pH elution buffer reached the column. If a protein was first loaded onto the column, no tryptic peptides of the protein were observed when trypsin was loaded later for the on‐column digestion. Only the autolysis peptides of trypsin were observed. On‐column digestion of 100 fmol myoglobin was successfully detected with a low sensitivity quadrupole mass spectrometer. A hybrid Os‐polymer/C₁₈ column tip was constructed for the online trypsin digestion of proteins in the aqueous buffers and the following trapping and elution of peptides from the C₁₈ column. The digestion of reduced and alkylated bovine serum albumin and human transferrin in 2.5 mM ammonium bicarbonate and 0.2 M urea buffer was observed on the column, with more peptide coverage than conventional 4 h in‐solution digestion at 37°C. Control experiments without the Os‐polymer in the column tip excluded the spontaneous in‐solution digestion of proteins in the short time window of buffer delivery onto the column, indirectly confirming the contribution of Os‐polymer on the fast trypsin digestion. Copyright © 2010 John Wiley & Sons, Ltd.     

Selective solid-phase isolation of methionine-containing peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine- and of methionine-sulfoxide-containing peptides

Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine- and methionine-sulfoxide-containing peptides, involving application of methionine-specific solid-phase reagent chemistry combined with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In the first step, methionine-containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine-sulfoxide-containing peptides and other methionine-free peptides are not bound and are washed out. The wash solution is used for MALDI-MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post-source decay (PSD) fragment ion analysis. Methionine-sulfoxide-containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2-mercaptoethanol at pH 8.5-8.8. The resulting peptides, mainly methionine-containing peptides, are analysed in a straightforward manner by MALDI-MS and seamless PSD. The strategy allows the fast identification of methionine- and methionine-sulfoxide-containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine-containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine-free peptides can be quite strongly bound non-covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that oxidation of some methionine residues occurred to a minor extent during the solid-phase isolation steps.     

Selective sampling of multiply phosphorylated peptides by capillary electrophoresis for electrospray ionization mass spectrometry analysis

The ionization of phosphorylated peptides in positive ion mode mass spectrometry is generally less efficient compared with the ionization of their non-phosphorylated counterparts. This can make phosphopeptides much more difficult to detect. One way to enhance the detection of phosphorylated proteins and peptides is by selectively isolating these species. Current approaches of phosphopeptide isolation are based on the favorable interactions of phosphate groups with immobilized metals. While these methods can be effective in the extraction, they can lead to incomplete sample recovery, particularly for the most strongly bound multiply phosphorylated components. A non-sorptive method of phosphopeptide isolation using capillary electrophoresis (CE) was recently reported [Zhang et al., Anal. Chem. 77 (2005) 6078]. The relatively low isoelectric points of phosphopeptides cause them to remain anionic at acidic sample pH. Hence, they can be selectively injected into the capillary by an applied field after the electroosmotic flow (EOF) is suppressed. The technique was previously coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, the exploitation of selective sampling in conjugation with electrospray ionization mass spectrometry (ESI-MS) is presented. The transition was not immediately straightforward. A number of major alterations were necessary for ESI interfacing. These adaptations include the choice of a suitable capillary coating for EOF control and the incorporation of organic solvent for efficient ESI. As expected, selective injection of phosphopeptides greatly enhanced the sensitivity of their detection in ESI-MS, particularly for the multiply phosphorylated species that were traditionally most problematic. Furthermore, an electrophoretic separation subsequent to the selective injection of the phosphopeptides was performed prior to analysis by ESI-MS. This allowed us to resolve the multiply phosphorylated peptides present in the samples, predominantly based on the number of phosphorylation sites on the peptides.     

Utilizing the inherent electrolysis in a chip-based nanoelectrospray emitter system to facilitate selective ionization and mass spectrometric analysis of metallo alkylporphyrins

A commercially available chip-based infusion nanoelectrospray ionization system was used to ionize metallo alkylporphyrins for mass spectrometric detection and structure elucidation by mass spectrometry. Different ionic forms of model compounds (nickel (II), vanadyl (II), copper (II), and cobalt (II) octaethylporphyrin) were created by using two different types of conductive pipette tips supplied with the device. These pipette tips provide the conductive contact to solution at which the electrolysis process inherent to electrospray takes places in the device. The original unmodified, bare carbon-impregnated plastic pipette tips were exploited to intentionally electrochemically oxidize (ionize) the porphyrins to form molecular radical cations for detection. Use of modified pipette tips, with a surface coating devised to inhibit analyte mass transport to the surface or slow the kinetics of the analyte electrochemical reactions, was shown to limit the ionic species observed in the mass spectra of these porphyrins largely, but not exclusively, to the protonated molecule. Under the conditions of these experiments, the effective upper potential limit for oxidation with the uncoated pipette tip was 1.1 V or less, and the coated pipette tips effectively prevented the oxidation of analytes with redox potentials greater than about 0.25 V. Product ion spectra of either molecular ionic species could be used to determine the alkyl chain length on the porphyrin macrocycle. The utility of this electrochemical ionization approach for the analysis of naturally occurring samples was demonstrated using nickel geoporphyrin fractions isolated from Gilsonite bitumen. Acquiring neutral loss spectra as a means to improve the specificity of detection in these complex natural samples was also illustrated.     

Enhanced Multi-phosphopeptide Enrichment and Nano LC-ESI-qTOF-MS Detection Strategy Using Click OEG-CD Matrix

Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Click OEG-CD[olio(ethylene glycol)(OEG) linked β-cyclodextrin(CD)] matrix has shown excellent performance in phosphopeptide enrichment. However, few multi-phosphopeptides were detected previously. In this investigation, an improved method aiming at enhancing the enrichment selectivity and mass spectrometry(MS) detection of multi-phosphopeptide was developed via optimizing the sample loading amount on per mg of matrix. Mass spectra were obtained on a Nano liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qTOF-MS). The enrichment selectivity of double-phosphopeptide could be enhanced with the increase of loading amount on per mg of matrix when taking the mixture of mono-, double-, and non-phosphopeptides as probe. Furthermore, the multi-phosphopeptide enrichment selectivity was enhanced under the condition of optimized loading amount when taking the product of typtic digestion α-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 α-casein phosphopeptide signals(including 15 multi-phosphopeptides) were detected. The result is much better than that of our previous report. The reduction of ion-suppression effect arised from the existence of high-abundance non-or mono-phosphopeptides and the stronger interactions between multi-phosphopepides and the matrix were contributed to the result. The study could be helpful to the better utilization of Click OEG-CD matrix in the enrichment of multi-phosphopeptide from complex biosamples in the subsequent investigation.     

Sample preparation and direct electrospray ionization on a tip column for rapid mass spectrometry analysis of complex samples

A handheld pipette tip column electrospray ionization source (PTC-ESI source) was developed for rapid mass spectrometry analysis at ambient pressure. The PTC-ESI source was made up of three main component parts including a micro DC high voltage (HV) power supply, a micropipette and a disposable micropipette tip filled with a plug of adsorbent. A DC high voltage was applied to the sharp point of the micropipette tip column to induce electrospray ionization. The PTC-ESI source was successfully used for direct analysis of basic organic compounds, organic acids and peptides in a simple matrix. In the case of complex samples, micro-extraction based on the adsorbent phase filled in the pipette tip was used to remove impurities and concentrate target analytes prior to ionization. The eluting solution was not pipetted out, but directly dispersed in the form of electrospray from the pipette tip for ionization. The effectiveness of the PTC-ESI source has been further demonstrated by fast analysis of therapeutic compounds and endogenous bioactive chemicals in complex biological samples.     

An evaluation of the utility of in vacuo methylation for mass-spectrometry-based analyses of peptides

In vacuo trimethylation of the N-terminus of a lyophilized peptide with methyl iodide was previously reported to enhance the peptide's signal in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and to suppress alkali adduct formation in electrospray ionization mass spectrometry (ESI-MS). Both the signal enhancement and alkali adduct suppression observed for methylated peptides are believed to be due to the permanent positive charge on the N-terminus of the peptide resulting from the formation of a quaternary ammonium moiety. The present work evaluates the general utility of the in vacuo methylation procedure for the MS analysis of peptides, and specifically addresses the issue of whether the methylation of nucleophilic sites other than the N-terminal amine affects the MALDI signal of modified peptides. This work establishes that, although certain side-chain modifications are inevitable using present reaction conditions, the derivatization leads to significant MALDI-MS signal improvement. The experimental results demonstrate that the N-terminal trimethylammonium derivatives of peptides exhibit MALDI signals comparable to or exceeding those of arginine-containing standards such as angiotensin I. The advantages and limitations of the in vacuo methylation procedure are discussed.