Latest developments in sample treatment for O-isotopic labeling for proteomics mass spectrometry-based approaches: A critical review

Nowadays isotopic ¹⁸O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using ¹⁸O-labeling. In this critical review we comment on the different ways in which ¹⁸O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage.     

A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis

In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and α-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test).     

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合¹⁸O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和¹⁸O标记效率,并对QconCAT蛋白质结合¹⁸O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。     

Accelerated 18O-labeling in urinary proteomics

Proteolytic (18)O-labeling of peptides has been studied and optimized in order to improve the labeling efficiency and to accelerate the process without increasing the degree of incomplete labeling. Using peptides generated from tryptic digested bovine serum albumin (BSA) and cytochrome c as model proteins, it was shown that complete labeling was achieved after 2 h at pH 6. To increase the sample throughput in a bottom-up proteomic setup, tryptic digestion of proteins in-solution was replaced with tryptic digestion using immobilized trypsin. As a result, an integrated approach was made possible, where both digestion (pH 8) and (18)O/(16)O-labeling of the resulting peptides (pH 6) were done using immobilized trypsin beads. This simplified the sample handling and reduced the overall reaction time significantly: the setup enabled tryptic digestion and (18)O/(16)O-labeling without sample transfer steps within 3.5 h with average (18)O/(16)O-ratios of 0.96±0.13 in aqueous buffer. The initial results were confirmed with a more complex matrix, by spiking urine with the model proteins, yielding results comparable with the ratios obtained in buffer. Satisfying ratios were also achieved regarding urinary proteins identified in a full scale bottom-up experiment. Average (18)O/(16)O-peptide ratios of 0.83±0.13 and 0.91±0.27 indicated good performance in a highly relevant matrix for biomarker discovery.     

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification

The relative quantification and identification of proteins by matrix‐assisted laser desorption ionization time‐of‐flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N‐terminal derivatization with 4‐sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while ¹⁸O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4‐sulphophenyl isothiocyanate derivatization with ¹⁸O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R² value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.     

A Bayesian Markov-Chain-Based Heteroscedastic Regression Model for the Analysis of <Superscript>18</Superscript>O-Labeled Mass Spectra

To reduce the influence of the between-spectra variability on the results of peptide quantification, one can consider the ¹⁸O-labeling approach. Ideally, with such labeling technique, a mass shift of 4 Da of the isotopic distributions of peptides from the labeled sample is induced, which allows one to distinguish the two samples and to quantify the relative abundance of the peptides. It is worth noting, however, that the presence of small quantities of ¹⁶O and ¹⁷O atoms during the labeling step can cause incomplete labeling. In practice, ignoring incomplete labeling may result in the biased estimation of the relative abundance of the peptide in the compared samples. A Markov model was developed to address this issue (Zhu, Valkenborg, Burzykowski. J. Proteome Res. 9, 2669–2677, 2010). The model assumed that the peak intensities were normally distributed with heteroscedasticity using a power-of-the-mean variance funtion. Such a dependence has been observed in practice. Alternatively, we formulate the model within the Bayesian framework. This opens the possibility to further extend the model by the inclusion of random effects that can be used to capture the biological/technical variability of the peptide abundance. The operational characteristics of the model were investigated by applications to real-life mass-spectrometry data sets and a simulation study.     

Simultaneous quantification and identification using <Superscript>18</Superscript>O labeling with an ion trap mass spectrometer and the analysis software application “ZoomQuant”

Stable isotope labeling with (18)O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of (18)O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers. In this paper, we describe a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using(18)O. The method utilizes zoom scans to provide high resolution data. This allows for accurate calculation of (18)O/(16)O ratios for peptides even when as much as 50% of a (18)O labeled peptide is present as the singly labeled species. The use of zoom scan data also provides sufficient resolution for calculating accurate ratios for peptides of +3 and lower charge states. Sequence coverage is comparable to that obtained with data acquisition modes that use only MS and MS/MS scans. We have employed a newly developed analysis software tool, ZoomQuant, which allows for the automated analysis of large data sets. We show that the combination of zoom scan data acquisition and analysis using ZoomQuant provides calculation of isotopic ratios accurate to approximately 21%. This compares well with data produced from (18)O labeling experiments using time of flight (TOF) and Fourier transform-ion cyclotron resonance (FT-ICR) MS instruments.     

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions

Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of ∼30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential ¹⁵N/¹⁴N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.     

Pd(0) or Pd(II)-catalyzed ring-opening reactions of benzylidene- and alkylidenecyclopropyl ketones and aldehydes

Pd(0) catalyzed reactions of methylenecyclopropyl carbonyl compounds afforded a convenient method for the synthesis of conjugate (E,E)-1,3-diene derivatives 2 in good to excellent yields. Moreover, we also found that Pd(II)-catalyzed reactions of methylenecyclopropyl carbonyl compounds with water gave 1,5-diketones in good to high yields via a carbene-palladium intermediate. The plausible reaction mechanisms have also been provided on the basis of control and ¹⁸O-labeling experiments.     

Study of Bulk Membrane Transport of Some Heavy Metal Cations Using Three Macrocyclic Ligands Containing Oxygen and Nitrogen Heteroatoms

The transport experiments of Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ag⁺ and Pb²⁺ metal cations were carried out by dibenzo-18-crown-6 (DB18C6), dibenzyl-diaza-18-crown-6 (Dibenzyl-diaza-18C6) and di-tert-butyl-dibenzo-18-crown-6 (Di-tert-butyl-DB18C6) using chloroform (CHCl₃), 1,2-dichloroethane (1,2-DCE) and nitrobenzene (NB) organic solvents as liquid membranes. The source phase contained equimolar concentration of these metal cations and the source and receiving phases being buffered at pH=5 and pH=3, respectively. The obtained results show that the selectivity and the efficiency of transport for these heavy metal cations change with the nature of the ligand and also the organic solvents, which were used as liquid membranes in these experiments. A good selectivity was observed for silver (I) ion by dibenzyl-diaza-18C6 in all membrane systems. Dibenzo-18C6 and di-tert-butyl-DB18C6 showed the highest transport efficiency for cobalt (II) ion. The effect of stearic acid on transport efficiency was also investigated and the results show that the efficiency of transport of the heavy metal cations increases in the presence of this organic acid.     

Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based techniques

We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on ¹⁸O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant ¹⁸O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that ¹⁸O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.     

A Polarographic Study of Tl+, Pb2+ and Cd2+ Complexes with Aza-18-crown-6 and Dibenzopyridino-18-crown-6 in some Binary Mixed Non-aqueous Solvents

The complexation of Tl⁺, Pb²⁺and Cd²⁺ cations by macrocyclic ligands, aza-18-crown-6 (L1) and dibenzopyridino-18-crown-6 (L2) was studied in some binary mixtures of methanol (MeOH), n-propanol (n-PrOH), nitromethane (NM) and acetonitrile (AN) with dimethylformamide (DMF) at 22 °C using DC (direct current) and differential pulse polarographic techniques (DPP). The stoichiometry and stability constants of the complexes were determined by monitoring the shifts in half-waves or peak potentials of the polarographic waves of metal ions against the ligand concentration. In all of the solvent systems, the stability of the resulting 1:1 complexes was found to be L1 > L2. The selectivity order of the L2 ligand for the cations was found to be Pb²⁺ > Tl⁺ > Cd²⁺ and the selectivity of the L1 ligand for Pb²⁺ ion was greater than that of Tl⁺ ion. The results show that the stability of the complexes depends on the nature and composition of the mixed solvents. There is an inverse relationship between the stability constants of the complexes and the amount of dimethylformamide in the mixed solvent systems.     

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

A Proton NMR Study of the Stoichiometry and Stability of 18-Crown-6 Complexes with K+, Rb+ and Tl+ Ions in Binary Dimethyl Sulfoxide-Nitrobenzene Mixtures

Proton NMR spectroscopy was used to study the complexation reaction of 18-crown-6 (18C6) with K⁺, Rb⁺ and Tl⁺ ions in a number of binary dimethyl sulfoxide-nitrobenzene mixtures. In all cases, the exchange between free and complexed crowns was fast on the NMR time scale and only a single population average ¹H signal was observed. Formation constants of the resulting 1:1 complexes in different solvent mixtures were determined by computer fitting of the chemical shift-mole ratio data. There is an inverse relationship between the complex stability and the amount of dimethyl sulfoxide in the mixed solvent. It was found that, in all solvent mixtures used, Rb⁺ ion forms the most stable complex with 18-crown-6 in the series.     

ON THE FORMATION AND REACTIVITY OF DIOXIRANE INTERMEDIATES IN THE REACTION OF PEROXOANIONS WITH ORGANIC SUBSTRATES*

Abstract— Kinetics and ¹⁸O-labeling studies have provided evidence for the involvement of dioxirane intermediates (V) in the ketone-catalysed decomposition of peroxomonosulfate (Caroate) HSO;. Reaction rates depend on ketone structure. In competition with catalysis of peroxide decomposition, the dioxirane intermediate is capable of oxidizing several organic and inorganic substrates. Thus, cis- and rrans-cinnammic acids could be converted stereospecifically into the corresponding epoxides. Also, oxidation of phenylpropiolic acid, a substrate which is representative of weakly nucleophilic alkynes, could be carried out using the Caroatehetone oxidizing system. Under the conditions adopted, no oxidation of the substrates examined was found to occur in the absence of ketone. The possibility that formation of dioxathiirane intermediates (XXII) occurs following a side pathway in the reaction of Caroate with sulfoxides (which produces sulfones in high yield) has been explored. Preliminary experiments using ¹⁸O-labeling of p-tolyl phenylsulfoxide, however. failed to support this hypothesis. pointing out the need for further detailed studies.     

Toward milk speciation through the monitoring of casein proteotypic peptides

The possibility of detecting extraneous milk in singles species cheese‐milk has been explored. A mass spectrometry (MS)‐based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the α_s1‐casein (CN) f8‐22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4‐22 peptide was selected as a marker for the two caprine α_s1‐CN A and B variants, which differ by a Pro¹⁶ (B)‐>Leu¹⁶ (A) substitution. MALDI analysis of the digest allowed the detection of α_s1‐CN f8‐22 and caprine α_s1‐CN f4‐22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)‐MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the α_s1‐CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI‐MS method. The isotopic‐label‐free quantification of isoform‐ or variant‐specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Screening for Protein Phosphorylation Using Nanoscale Reactions on Microdroplet Arrays

We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano‐LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×?80 Da. The MALDI‐MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions.     

The reaction of mono-aryl substituted methylenecyclobutanes with diphenyl diselenide in the presence of iodosobenzene diacetate and H2O

The mono-aryl substituted methylenecyclobutanes undergo an interesting reaction with diphenyl diselenide in the presence of iodosobenzene diacetate and H₂O at 40 °C in 1,2-dichloroethane to give the corresponding aryl-(1-phenylselanylcyclobutyl)methanones in moderate to good yields within 30 h. A plausible reaction mechanism has been discussed on the basis of the control and ¹⁸O-labeling experiments.