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A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.
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PMID:Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry. 1119 5

Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS.
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PMID:Determination of phosphorus in small amounts of protein samples by ICP-MS. 1261 Jul 11

A comparison of different nebulisers for direct hyphenation of capillary and nano liquid chromatography (Cap-LC, Nano-LC) and quadrupole-based collision cell inductively coupled plasma mass spectrometry (CC-ICP-MS) for phosphorylation profiling of tryptic protein digests is described. Helium was used as cell gas and specially tuned instrumental conditions were used to achieve background minimisation at the mass of phosphorus, because of kinetic energy discrimination of the interfering polyatomic ions. The proposed set-up is based on a modified capillary electrophoresis interface and a home-made 4 mL spray chamber. It enables the use of gradient conditions with a highly concentrated organic mobile phase as often used in protein phosphorylation analysis, without the need to apply membrane desolvation for removal of the organic phase or further background minimisation. No significant signal suppression or other negative effects caused by the organic mobile phase occur, because of the low flow rates used in Cap-LC and the robust plasma conditions of the CC-ICP-MS instrument. A tryptic digest of beta-casein was investigated as model compound to demonstrate the applicability of the proposed set-up for phosphorylation profiling in protein analysis using quadrupole based collision-cell ICP-MS as phosphorus-specific detector. Detection limits for phosphorylated peptides down to the sub picomole level were obtained. As a complementary technique, electrospray ionisation tandem mass spectrometry (ESI-MS-MS) with data base searching was used for further characterisation of the phosphorylated peptides detected.
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PMID:Development and characterisation of a new interface for coupling capillary LC with collision-cell ICP-MS and its application for phosphorylation profiling of tryptic protein digests. 1559 18

The validity of using elemental phosphorus standards to accurately and precisely quantify phosphopeptides by capillary HPLC (capHPLC) coupled to ICP-collison cell-MS is investigated in detail. Operating requirements to maintain stable (31)P sensitivity along the reversed-phase gradient are described. Specifically, the use of an optimum postcolumn makeup flow with a defined acetonitrile content turned out to be necessary to buffer the acetonitrile variation of the capillary chromatographic eluent and ensure plasma stability. Then, a highly pure P-containing standard (bis(4-nitro-phenyl) phosphate, BNPP) was spiked into the samples and used to quantify them with very low absolute errors (2-4%) and excellent precision (3-6%). The capHPLC-ICPMS method showed excellent linearity over 3 orders of magnitude and provided adequate detection limits (110 fmol, 3.4 pg P). Accurate quantification of the phosphopeptides present in a tryptic digest of beta-casein and casein from bovine milk was then attempted. Previously, and in order to be able to close mass balances, total P contents, percentages of inorganic P present, and recoveries from the reversed-phase column used in the separation were computed for each sample. Quantification using the spiked BNPP for the different phosphopeptides detected matched the expected values well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidating amino acid sequences, a requisite still necessary to translate the determined amount of P in each chromatographic peak into amount of phosphopeptide. The great potential of these strategies, based on ICPMS detection, to assess the many procedures proposed and commonly used for purification, preconcentration, and/or isolation of phosphopeptides in phosphoproteomics studies is demonstrated using a commercially available titanium dioxide (TiO(2)) cartridge for phosphopeptide enrichment from complex mixtures. Quantitative results obtained allow one to assess individual phosphopeptide recoveries from the TiO(2) cartridge with unsurpassed accuracy. Of course, this information is essential for reliable absolute quantifications in phosphoproteomics.
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PMID:Absolute and site-specific quantification of protein phosphorylation using integrated elemental and molecular mass spectrometry: its potential to assess phosphopeptide enrichment procedures. 1824 85

Molecular mass spectrometry (MS) analysis of protein phosphorylation is partially limited by the molecular specie specificity of the analytical responses that might impair both qualitative and quantitative performances. Elemental MS, such as inductively coupled plasma mass spectrometry (ICP-MS) can overcome these drawbacks; in fact, analytical performance is theoretically independent of the molecular structure of a target analyte naturally containing the elements of interest. Nevertheless, isobaric interferences derived from sample matrix and laboratory environment can hinder the quantitative determination of both phosphorus (P) and sulfur (S) as (31)P(+) and (32)S(+) by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) under standard plasma conditions. These interferences may be overcome by quantifying P and S as oxide ions (31)P(16)O(+) and (32)S(16)O(+), respectively. In this study, we present a systematic investigation on the effect of plasma instrumental conditions on the oxide ion responses by a design of experiment approach for the simultaneous ICP-QMS determination of P and S ((31)P(16)O(+) and (32)S(16)O(+), respectively) in protein samples without the use of dynamic reaction, collision reaction cells or pre-addition of oxygen as reactant gas in the torch. The proposed method was evaluated in terms of limit of detection, limit of quantification, linearity, repeatability, and trueness. Moreover, detection and quantification capabilities of the optimized method were compared to the standard plasma mode for determination of (31)P(+) and (34)S(+). Spectral and non-spectral interferences affecting the quantification of (31)P(+), (31)P(16)O(+) and (32)S(16)O(+) were also studied. The suitability of inorganic elemental standards for P and S quantification in proteins was assessed. The method was applied to quantify the phosphorylation stoichiometry of commercially available caseins (bovine beta-casein, native and dephosphorylated alpha-casein) and results were confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight MS analysis. We demonstrate that ICP-QMS, by quantifying P and S as oxide ions, was able to accurately calculate the degree of phosphorylation of beta-casein and alpha-casein and to detect specific partial enzymatic dephosphorylation. The collected results might lead to further development of ICP-QMS interfaces optimized for protein phosphorylation studies and for proteomics investigations.
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PMID:Protein phosphorylation stoichiometry by simultaneous ICP-QMS determination of phosphorus and sulfur oxide ions: a multivariate optimization of plasma operating conditions. 2008 9

This study describes a modification to tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis to make it more effective for the separation of low molecular mass proteins and for coupling with inductively coupled plasma mass spectrometry (ICP-MS). The modified method employs low-percentage polyacrylamide gels (7-10%) (w/v) and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. Using phosphopeptides as a model system, and offline analysis, we obtained recoveries of 73% (w/v) in a 9% gel compared with 55% in a conventional 16% gel. Online coupling was achieved by modification of a standard commercially available gel electroelution apparatus and casting of the gel into a 7.3-cm-long tube. Online separation of a digest of beta-casein was demonstrated with recovery of the mono- and tetraphosphopeptides, which were identified by comparison with peptide standards. A mass balance study with the standards yielded recoveries of 95% for tetraphosphopeptides and 48% for monophosphopeptides. The factors affecting the separations and recoveries are discussed in detail. The detection limits for 10-microL samples of the mono- and tetraphosphopeptides were 0.7 microM (7 pmol) and 0.2 microM (2 pmol) respectively.
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PMID:Modification of tricine-SDS-PAGE for online and offline analysis of phosphoproteins by ICP-MS. 2022 54