Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.     

Preparation of monodisperse immobilized Ti affinity chromatography microspheres for specific enrichment of phosphopeptides

This study presented an approach to prepare monodisperse immobilized Ti⁴⁺ affinity chromatography (Ti⁴⁺-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti⁴⁺ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti⁴⁺-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti⁴⁺-IMAC microspheres were compared with commercial Fe³⁺-IMAC adsorbent and homemade Zr⁴⁺-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe³⁺-IMAC, Zr⁴⁺-IMAC and Ti⁴⁺-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti⁴⁺-IMAC had the best performance for enrichment of phosphopeptides.     

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.     

Highly efficient enrichment of phosphopeptides by magnetic nanoparticles coated with zirconium phosphonate for phosphoproteome analysis

The location of phosphorylation plays a vital role for the elucidation of biological processes. The challenge of low stoichiometry of phosphoproteins and signal suppression of phosphopeptides by nonphosphopeptides in mass spectrometry (MS) analysis makes the selective enrichment of phosphopeptides prior to MS analysis necessary. Besides the immobilized metal affinity chromatography (IMAC) method, some affinity methods based on nanoparticles displayed a higher enrichment efficiency for phosphopeptides such as Fe(3)O(4)/TiO2 and Fe(3)O(4)/ZrO(2) nanoparticles. To further improve the selectivity and compatibility of the affinity methods, a novel strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides has been developed in this study. Under optimized experimental conditions, 1 x 10(-9) M phosphopeptides in 50 microL tryptic digest of beta-casein could be enriched and identified successfully. Reliable results were also obtained for 1 x 10(-8) M phosphopeptides in 50 microL tryptic digest of beta-casein in the presence of nonphosphopeptides from a tryptic digest of bovine serum albumin (BSA) over 20 times in concentration. The performance of nanoparticles for use in a real sample was further demonstrated by employing the strong cation-exchange chromatography (SCX) fraction of a tryptic digest of a protein extract from Chang liver cells as a model sample. Experimental results show that the nanoparticles can be easily and effectively used for enrichment of phosphopeptides in low concentration. Most importantly, our approach is more compatible with commonly used SCX strategies than Fe(3+)-IMAC. The proposed method thus has great potential for future studies of large-scale phosphoproteomes.     

Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate metal ion interactions with salivary peptides histatin 3 (H3) and histatin 5 (H5). Conformational changes of these peptides in the presence of metal ions were studied using circular dichroism spectroscopy. H3 and H5 formed high affinity complexes with Cu(2+) and Ni(2+) and, to a lesser extent, with Zn(2+). Both peptides show the potential for multiple binding sites for Cu(2+) and Ni(2+) and only a single strong binding site for Zn(2+). The binding of a third Cu(2+) ion to H3 seems to enable the binding of a fourth ion to H3. The binding of a second and third Ni(2+) ion to H5 has a similar effect in enabling the binding of a fourth ion. None of the metal ions examined stabilized a regular secondary structure for either peptide. Subtle changes in overall conformation are seen with the addition of Cu(2+) to both H3 and H5.     

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.     

基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用

磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti⁴⁺)螯合IMAC材料(Ti⁴⁺-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti⁴⁺-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti⁴⁺-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti⁴⁺-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。     

Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example.     

Selective enrichment of endogenous peptides by chemically modified porous nanoparticles for peptidome analysis

We report the development of a combined strategy for high capacity, comprehensive enrichment of endogenous peptide from complex biological samples at natural pH condition. MCM-41 nanoparticles with highly ordered nanoscale pores (i.e. 4.8nm) and high-surface area (i.e. 751m(2)/g) were synthesized and modified with strong cation-exchange (SCX-MCM-41) and strong anion-exchange (SAX-MCM-41) groups. The modified nanoparticles demonstrated good size-exclusion effect for the adsorption of standard protein lysozyme with molecular weight (MW) of ca. 15kDa; and the peptides with MW lower than this value can be well adsorbed. Step elution of the enriched peptides with five salt concentrations presented that both modified nanoparticles have high capacity and complementarity for peptides enrichment, and the SAX-MCM-41 nanoparticles has obviously high selectivity for acidic peptides with pI (isoelectric point) lower than 4. Large-scale enrichment of endogenous peptides in 2mg mouse liver extract was achieved by further combination of SCX-MCM-41 and SAX-MCM-41 with unmodified MCM-41 nanoparticles. On-line 2D nano-LC/MS/MS was applied to analyze the enriched samples, and 2721 unique peptides were identified in total. Two-dimensional analysis of MW versus pI distribution combined with abundance of the identified peptides demonstrated that the three types of nanoparticles have comprehensive complementarity for peptidome enrichment.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.     

An unusual cleavage reaction of a peptide observed during dithiotreitol and tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry

A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.     

Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses

Several affinity resins consisting of ionic metals or metal oxides were investigated for their phosphopeptide enrichment capabilities with subsequent mass spectrometric analyses. Commercially-available enrichment metal oxide affinity chromatography (MOAC) resins using manufacturer’s and/or published protocols were compared and evaluated for the most efficient and selective method that could be implemented as a standard enrichment procedure. From these comparative analyses, using a tryptic digest of casein proteins, it was determined that in our hands, two of the resins out-performed the others based on a variety of criteria, including the number of phosphorylation sites identified during MS analyses, the lower numbers of nonspecifically bound peptides observed, and the limits of detection. Applicability of these enrichment resins to a complex biological mixture was investigated. For this work, a mixture of avian histones was digested, subjected to titanium dioxide phosphopeptide enrichment, and analyzed by mass spectrometry. Eight phosphorylated tryptic peptides were observed following enrichment and subsequent LC/MS/MS analyses. Of note, seven of the eight phosphopeptides were not observed without titanium dioxide enrichment. From these analyses, four sites of phosphorylation were unequivocally determined, two of which have not been reported previously. Four additional phosphopeptides were observed; however, the site of phosphorylation could not be distinguished but was localized to one of two possible amino acids. These methods should aid in the investigation of proteins post-translationally modified with phosphate, especially those present at low concentrations as was demonstrated by successful enrichment at the femtomole level.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义。高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展。人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤。为了研究样品制备方法对分析结果的影响,分别采用不变性超滤法(UF 1)、热变性超滤法(UF 2)、化学变性超滤法(UF 3)、三氯乙酸沉淀法(PCPN 1)、乙醇沉淀法(PCPN 2)、强疏水性碳介孔材料(highly ordered mesoporous carbon, OMC)富集法等6种方法从人乳中提取内源肽,利用LC-MS/MS研究样品制备方法对人乳内源肽分析结果的影响。结果表明,UF 1和UF 2法制备的样品中可鉴定到的肽段数目分别为1161±8条和1017±91条,两种方法制备的样品中肽序列的重合率大于70%, UF 1在所有方法中鉴定到的肽的数目最多。UF 3法制备的样品所能鉴定到的肽段数目最少,仅为366±18条。PCPN 1和PCPN 2两种沉淀法制备样品中的内源肽分别为779±69和876±55条,但内源肽差异较大,仅有约50%肽段序列重合。OMC法制备样品中肽的数目为549±151条,与其他方法相比,虽然鉴定的肽数量上没有优势,但该方法制备的样品中肽在等电点(pI)和亲水性平均系数(GRAVY)等性质上没有偏倚,说明该法可用于制备特定人乳内源肽。6种方法制备的样品中鉴定到来源于β-酪蛋白、免疫球蛋白受体、骨桥蛋白、α_S1-酪蛋白、κ-酪蛋白和胆盐激活脂肪酶的肽,并且源于以上蛋白质的肽段总数在该样品中均超过88%,说明6种方法制备的样品都可以满足鉴定一般人乳内源肽的需求。UF 2、UF 3和OMC法制备的样品中鉴定到源于乳铁蛋白的内源肽的数目分别为21、38和19条,内源肽在乳铁蛋白上的覆盖率分别为14%、16%和19%,而文献常用的PCPN 1法制备的样品则会丢失此类内源肽。综上,UF 2法制备的样品不仅肽段数量多、母体蛋白质种类丰富,还可鉴定到源于乳铁蛋白的肽,可作为人乳内源肽组学研究中的首选方法。     

Fe3O4@Al2O3 magnetic core-shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres over Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC (immobilized metal affinity chromatography) resin, and TiO(2) beads. Finally, the Al(2)O(3) coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.     

Polythiol binding to biologically relevant metal ions

The coordination properties of three peptides with CXXC motif: Ac-GCASCDNCRACKK-NH(2), Ac-GCASCDNCRAAKK-NH(2) and Ac-GCASCDNARAAKK-NH(2) as donors of four, three and two thiol ligands for Ni(2+),Cd(2+), Zn(2+) and Bi(3+) were studied by potentiometric titrations, UV-Vis and CD spectra measurements. Since the stability of the complexes is closely connected with the amount of the metal-bound cysteine sulfurs, competition plots of the complexes of peptides with 2, 3 and 4 cysteines further prove the involvement of all thiols in the metal ion binding. Furthermore, the sulfur-bound zinc complexes appear to be much more stable than the sulfur-bound nickel ones. The stabilities of the studied complexes decreases in the series Bi(3+) ? Cd(2+) > Zn(2+) > Ni(2+).     

Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere

In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan–glycidyl methacrylate (GMA)–iminodiacetic acid (IDA)–Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe³⁺-IMAC and TiO₂, these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe³⁺ nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS.     

Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe₃O₄) and titanium dioxide (TiO₂) particles, to detect mono‐ and multi‐phosphorylated peptides. In the first step, phosphopeptides were captured on Fe₃O₄ particles. In a subsequent step, any residual phosphopeptides were captured on TiO₂ particles. The particles were eluted and rinsed to yield phosphopeptide‐enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe₃O₄/TiO₂ enrichment strategy was demonstrated by the successful enrichment of bovine α‐casein phosphopeptides. We then applied the sequential Fe₃O₄/TiO₂ enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono‐ and multi‐phosphorylated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

Observation of an unusually facile fragmentation pathway of gas-phase peptide ions: a study on the gas-phase fragmentation mechanism and energetics of tryptic peptides modified with 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC) and their 18-crown-6 complexes

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.     

Preconcentration and determination of lead, cadmium and nickel from water samples using a polyethylene glycol dye immobilized on poly(hydroxyethylmethacrylate) microspheres.

A simple and sensitive preconcentration analysis-atomic absorption spectrometric procedure is described for the determination of lead, cadmium and nickel. The method is based upon on-line preconcentration of metal ions on a minicolumn of Cibacron Blue F3-GA immobilized on poly(hydroxyethylmethacrylate), poly(HEMA). The enrichment factors obtained were 42 for lead, 52 for cadmium and 63 for nickel (sample volume 10 mL and sample flow rate 5 mL/min). The relative standard deviations (n = 10), in 10 mL sample solutions containing 100 microg/L Pb(2+), 10 microg/L Cd(2+) and 100 microg/L Ni(2+) were 8.9, 3.7 and 3.5%, respectively. The limits of detection (blank + 3s) (n = 10), were found to be 12.01 microg/L for Pb(2+), 1.34 microg/L for Cd(2+) and 28.73 microg/L for Ni(2+). The accuracy of the system was checked with certified and tap water samples spiked with known amounts of metal ions. No significant difference was found between the achieved results and the certified values.