Protein phosphorylation is one of the most important posttranslational modifications that is critically involved in many significant cellular processes.[1] It is estimated that one third of all proteins in eukaryotic cells are phosphorylated at any given time.[2] Moreover, a single protein can be phosphorylated and dephosphorylated by different kinases and phosphatases, respectively, on different sites at different times. These phosphorylation variations, that is, resulting from a targeted perturbation, can only be detectable if quantitative information is available. Unfortunately, quantification of protein phosphorylation is a very challenging task that is often hampered by low relative amounts of phosphoproteins and a lack of adequate analytical methods.[3] ESI and MALDI molecular mass spectrometry have been successful in identifying and measuring relative changes in quantity of a particular (phospho)protein.[4] Element mass spectrometry (inductively coupled plasma, ICPMS) has also been reported to compute protein phosphorylation stoichiometry by using the relative measurement 31 P/ 34 S and the protein sequence information obtained by ESIMS. [5,6] As this latter approach requires the presence of S-containing residues (cysteine or methionine), it mostly provides the phosphorylation degree of the whole protein studied. Furthermore, sample-preparation steps, such as reduction and alkylation, may strongly affect the P/S ratio obtained, leading to biased protein phosphorylation results.Absolute quantification of phosphoproteins at given phosphorylation sites is much less commonly addressed, and so far reported methods require chemical synthesis (preferably with incorporated stable isotopes) of each individual phosphopeptide, which must already be known. [7,8] In fact, the main limiting factor to obtain absolute and reliable phosphorylation quantifications is the lack of the phosphopeptide and phosphoprotein standards required. Interestingly, the elemental response by ICPMS, when operated under certain conditions, could be directly proportional to the absolute amount of the element introduced (P in this case).[9] Therefore, in contrast to molecular MS techniques, the signal is independent of the species and sample matrix. However, a problem arises when ICPMS is used as an elemental detector in reversed-phase gradients in which the organic content (mostly acetonitrile) of the mobile phase strongly influences the ionization efficiency in the plasma, even at capillary (4 mL min À1 ) [10] and nano (300 nL min À1 ) [11] flow rates. Herein, we describe the addition of a postcolumn sheath flow with a constant acetonitrile content that is able to buffer gradient composition changes. This leads to a constant 31 P sensitivity along the mHPLC-ICPMS gradient, which is required to separate the different tryptic phosphopeptides originally present in the sample digest and the spiked Pcontaining standard. We then investigated the accuracy and precision that is attainable by using commercially available phosphopeptides. Moreover, th...

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.

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The validity of using tyrosine iodination chemistry for the absolute and generic quantification of peptides by capillary high-performance liquid chromatography (capHPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is investigated in detail. In this approach, two iodine atoms are specifically bioconjugated to the meta positions of the aromatic ring of every tyrosine residue. Characterization studies by capHPLC with parallel ICPMS and electrospray ionization tandem mass spectrometry (ESIMS/MS) detection clearly showed that such labeling iodination reaction affords one to obtain most accurate peptide determinations (after translation of the picomoles of iodine, quantified by ICPMS in each chromatographic peak, into picomoles of the corresponding labeled peptide). It is demonstrated that only, but every, tyrosine residue present in the peptide is completely diiodinated. The excellent detection limits for iodine using ICPMS allowed robust and highly sensitive tyrosine-containing peptide quantification (480 pM, 480 amol absolute). Derivatization is easily accomplished in a water/acetonitrile solution in only 2 min. Moreover, since the signal in ICPMS is completely independent from the chemical species containing the detected element, any iodine-containing standard (e.g., iodobenzoic acid) could be used as internal standard for the absolute quantification of every iodine-labeled tyrosine-containing peptide separated and detected along the gradient. The approach was optimized for tyrosine labeling and then validated by application to the absolute quantification of the three standard peptides present in the only reference material for peptide quantity (NIST 8327) commercially available. Identification of the species quantified by ICPMS was carried out by parallel capHPLC-ESI quadrupole time-of-flight (Q/TOF) analysis and corresponded, as expected, to the diiodinated peptides. The collision-induced dissociation (CID) spectra obtained demonstrated unequivocally the specific and complete derivatization of the tyrosine residues. The obtained quantitative results closely matched the reference values reported by the National Institute of Standards and Technology (NIST). In terms of precision, the relative standard deviation was as low as 3% RSD. Finally the approach was tested for the absolute quantification of proteins using a model standard protein (beta-casein). Results agreed again with the value specified showing that this labeling reaction is compatible with tryptic digestion.

Protein phosphorylation is one of the most important posttranslational modifications that is critically involved in many significant cellular processes.[1] It is estimated that one third of all proteins in eukaryotic cells are phosphorylated at any given time.[2] Moreover, a single protein can be phosphorylated and dephosphorylated by different kinases and phosphatases, respectively, on different sites at different times. These phosphorylation variations, that is, resulting from a targeted perturbation, can only be detectable if quantitative information is available. Unfortunately, quantification of protein phosphorylation is a very challenging task that is often hampered by low relative amounts of phosphoproteins and a lack of adequate analytical methods.[3] ESI and MALDI molecular mass spectrometry have been successful in identifying and measuring relative changes in quantity of a particular (phospho)protein.[4] Element mass spectrometry (inductively coupled plasma, ICPMS) has also been reported to compute protein phosphorylation stoichiometry by using the relative measurement 31 P/ 34 S and the protein sequence information obtained by ESIMS. [5,6] As this latter approach requires the presence of S-containing residues (cysteine or methionine), it mostly provides the phosphorylation degree of the whole protein studied. Furthermore, sample-preparation steps, such as reduction and alkylation, may strongly affect the P/S ratio obtained, leading to biased protein phosphorylation results.Absolute quantification of phosphoproteins at given phosphorylation sites is much less commonly addressed, and so far reported methods require chemical synthesis (preferably with incorporated stable isotopes) of each individual phosphopeptide, which must already be known. [7,8] In fact, the main limiting factor to obtain absolute and reliable phosphorylation quantifications is the lack of the phosphopeptide and phosphoprotein standards required. Interestingly, the elemental response by ICPMS, when operated under certain conditions, could be directly proportional to the absolute amount of the element introduced (P in this case).[9] Therefore, in contrast to molecular MS techniques, the signal is independent of the species and sample matrix. However, a problem arises when ICPMS is used as an elemental detector in reversed-phase gradients in which the organic content (mostly acetonitrile) of the mobile phase strongly influences the ionization efficiency in the plasma, even at capillary (4 mL min À1 ) [10] and nano (300 nL min À1 ) [11] flow rates. Herein, we describe the addition of a postcolumn sheath flow with a constant acetonitrile content that is able to buffer gradient composition changes. This leads to a constant 31 P sensitivity along the mHPLC-ICPMS gradient, which is required to separate the different tryptic phosphopeptides originally present in the sample digest and the spiked Pcontaining standard. We then investigated the accuracy and precision that is attainable by using commercially available phosphopeptides. Moreover, th...