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Triple Quad ICP-MS: Pushing the limits for quantitation of phosphorus and sulfur


Inductively coupled plasma mass spectrometry is an ideal tool to quantify elemental concentrations and in case of known stochiometry to quantify compounds. The main feature, allowing for such quantification is the general compound independent element response of the system hampered only by spectral interferences caused by polyatomic species. Quantification schemes used in proteomics mostly relies on the use of synthetic isotopically labeled peptides/proteins which are used as internal standards for the quantification of the corresponding targeted species. It is clear that sensitive and interference-free quantification of ICP-detectable elements naturally present in proteins will boost the role of ICP-MS in proteomics. Unfortunately, the determination of heteroatoms present in many proteins, namely sulfur (being present in about 5% in natural proteine) and phosphorus (because of phosphorylation in about 1/3 of all proteins in eukaryotic cells) is highly compromised by their high ionization potentials and further limited by serious polyatomic interferences. Existing approaches up to know relied on high resolution mass spectrometry using sector field instruments or reaction cells used in quadrupole instruments. Using such systems, absolute detection limits achieved are in the low pmol and medium-high fmol range for S- and P-containing species, respectively, still far from those reported using ESI-MS/MS and isotopically labeled synthetic peptides.

The new study:
A spanish research group headed by Alfredo Sanz-Medel now for the first time proposed a new concept of polyatomic interference removal in ICP-MS using tandem MS for S and P-containing species analysis.

Using a novel Triple Quad ICP-MS (Agilent 8800 ICPQQQ, Tokyo, Japan), oxygen (Ar:O2 mixture, 8:2) was introduced to the ICP at a flow rate of 0.08 L/min in order to oxidise the carbon in the plasma resulting from the introduction of the organic mobile phase (acetonitrile). Oxygen was also introduced in the cell at a flow rate of 0.35 mL min.

Fig. 1: ICP-QQQMS detection schemes for phosphorus and sulfur

Figure 1 depicts how the new triple-quad system deals with the serious polyatomic interferences suffered by S and P during ICP-MS analysis. The first quadrupole (Q1) works as 1 amu window band-pass mass filter selecting targeted analyte ions (P and S) and their polyatomic isobaric interferences. This is the first specificity level. Reaction with O2 in the cell separates analytes and interferences by shifting analytes to new product ion masses because reaction rate with O2 is much higher for 31P+ and 32S+ (and 34S+) than for their polyatomic interferences. This leads to interference-free detection of P and S after setting the second quadrupole (Q2) at 16 mass units higher than Q1: m/z 47 (PO+), 48 (32SO+), and 50 (34SO+). This is the second specificity level where polyatomic interferences at nominal masses of P (15N16O+, 14N16OH+, 14N17O+, 12C18OH+) and S (16O2+, 15N17O+, 15N16OH+, 14N18O+) are removed.

The main advantage of the new ICP-QQQ compared to an single quad instrument equipped with DRC (ICP-DRC-MS) is the first specificity level set at Q1. In fact, Q1 rejects all masses except analyte (and its on-mass interferences) ensuring that all the product ions analyzed in Q2 are derived from the selected precursor ion, reducing the background level.

Using this detection schemes, the new systen was optimized for the hyphenated mode with  capHPLC being operated at low flow (5 µL/min) and high organic mobile phase (30 % acetonitrile). Obtained detection limits were as low as 1.2 ng/mL for 32S and 0.6 ng/mL  for 31P and were mostly limited by blank values originating from contaminated organic reagents.

To demonstrate the feasibility of the new approach the researchers selected the determination of bis(4-nitro-phenyl)phosphate (BNPP)  and methionine as examples for P- and S-containing species. Linear calibration graphs were obtained for the working range of 0-200 ng/mL and limits of detection were 0.18 ng/mL S and 0.10 ng/mL P, which translate into 11 fmol and 6.6 fmol absolute, respectively. The authors state, that such good detection limits have not been achieved before, and are more than two orders of magnitude better for sulfur than reported with other systems.

The original study:

Silvia Diez Fernández, Naoki Sugishama, Jorge Ruiz Encinar, Alfredo Sanz-Medel, Triple Quad ICPMS (ICPQQQ) as a New Tool for Absolute Quantitative Proteomics and Phosphoproteomics, Anal. Chem., 84 (2012) 5851-5857. doi: 10.1021/ac3009516

Related studies:

Rui Liu, Yi Lv, Xiandeng Hou, Lu Yang, Zoltan Mester, Protein Quantitation Using Ru-NHS Ester Tagging and Isotope Dilution High-Pressure Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry Determination, Anal. Chem., 84/6 (2012) 2769–2775. DOI: 10.1021/ac203141d

Ana Pereira Navaza, Jorge Ruiz Encinar, Alfredo Ballesteros, José M. González, Alfredo Sanz-Medel, Capillary HPLC-ICPMS and Tyrosine Iodination for the Absolute Quantification of Peptides Using Generic Standards, Anal. Chem. 2009, 81, 5390–5399. doi: 10.1021/ac9005606

Nico Zinn, Bettina Hahn, Rüdiger Pipkorn, Dominik Schwarzer, Wolf D. Lehmann, Phosphorus-Based Absolutely Quantified Standard Peptides for Quantitative Proteomics, J. Proteome Res., 8/10 (2009) 4870–4875. DOI: 10.1021/pr900494m

Nico Zinn, Ralf Krüger, Peter Leonhard, Jörg Bettmer, µLC coupled to ICP–SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification, Anal. Bioanal. Chem., 391 (2008) 537–543. DOI: 10.1007/s00216-008-2025-9

Ana Pereira Navaza, Jorge Ruiz Encinar, Montserrat Carrascal, Joaquín Abián, Alfredo Sanz-Medel, Absolute and Site-Specific Quantification of Protein Phosphorylation Using Integrated Elemental and Molecular Mass Spectrometry: Its Potential To Assess Phosphopeptide Enrichment Procedures, Anal. Chem., 80 (2008) 1777-1787. 10.1021/ac7022316

Dirk Schaumlöffel, Pierre Giusti, Hugues Preud’Homme, Joanna Szpunar, Ryszard Lobinski, Precolumn Isotope Dilution Analysis in nanoHPLC-ICPMS for Absolute Quantification of Sulfur-Containing Peptides, Anal. Chem., 79 (2007) 2859-2868. doi: 10.1021/ac061864r

A. Pereira Navaza, J. Ruiz Encinar, A. Sanz-Medel, Absolute and Accurate Quantification of Protein Phosphorylation by Using an Elemental Phosphorus Standard and Element Mass Spectrometry, Angew. Chem., Int. Ed., 46 (2007) 569– 671. DOI: 10.1002/anie.200602517

Stefan Stürup, Lars Bendahl, Bente Gammelgaard, Optimization of LC-DRC-ICP-MS for the speciation of selenotrisulfides with simultaneous detection of sulfur and selenium as oxides combined with determination of elemental and isotope ratios,  J. Anal. At. Spectrom., 21/2 (2006) 201-203. DOI: 10.1039/B514633J

Daniel Pröfrock, Peter Leonhard, Wolfgang Ruck, Andreas Prange, 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, Anal. Bioanal. Chem., 381 (2005) 194–204. DOI 10.1007/s00216-004-2930-5

C.J. Smith, I.D. Wilson, L. Weidolf, F. Abou-Shakra, M. Thomsen, Enhanced Detection of Sulphur and Phosphorous Containing Compounds in HPLC-Inductively Coupled Plasma Mass Spectrometry Using Chemical Resolution via Hexapole-Based Reaction with Oxygen, Chromatographia,  Suppl. 59 (2004) S165-S170. Doi: 10.1365/s10337-004-0185-0

Stephan Hann, Gunda Koellensperger, Christian Obinger, Paul Georg Furtmüller, Gerhard Stingeder, SEC-ICP-DRCMS and SEC-ICP-SFMS for determination of metal–sulfur ratios in metalloprotein, J. Anal. At. Spectrom., 19 (2004) 74-79.  doi: 10.1039/b310650k

Daniel Pröfrock, Peter Leonhard, Andreas Prange, Determination of sulfur and selected trace elements in metallothionein-like proteins using capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell, Anal. Bioanal. Chem., 377 (2003) 132–139. DOI 10.1007/s00216-003-2041-8

Mathias Wind, Ansgar Wegener, Andreas Eisenmenger, Roland Kellner, Wolf D. Lehmann, Sulfur as the Key Element for Quantitative Protein Analysis by Capillary Liquid Chromatography Coupled to Element Mass Spectrometry, Angew. Chem. Int. Ed., 42 (2003) 3425–3427. DOI: 10.1002/anie.200250547

Daniel Pröfrock, Peter Leonhard, Andreas Prange, Determination of phosphorus in phosphorylated deoxyribonucleotides using capillary electrophoresis and high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell, J. Anal. At. Spectrom., 18 (2003) 708–713. DOI: 10.1039/b302445h

Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Reaction cells and collision cells for ICP-MS: a tutorial review, Spectrochimica Acta Part B, 57 (2002) 1361–1452. doi: 10.1016/S0584-8547(02)00069-1

Dmitry R. Bandura, Vladimir I. Baranov, Scott D. Tanner, Detection of Ultratrace Phosphorus and Sulfur by Quadrupole ICPMS with Dynamic Reaction Cell, Anal. Chem. 2002, 74, 1497-1502. doi: 10.1021/ac011031v

Mathias Wind, Michael Edler, Norbert Jakubowski, Michael Linscheid, Horst Wesch, Wolf D. Lehmann, Analysis of Protein Phosphorylation by Capillary Liquid Chromatography Coupled to Element Mass Spectrometry with 31P Detection and to Electrospray Mass Spectrometry, Anal. Chem. 2001, 73, 29-35. doi: 10.1021/ac0009595

Mathias Wind, Horst Wesch, Wolf D. Lehmann, Protein Phosphorylation Degree: Determination by Capillary Liquid Chromatography and Inductively Coupled Plasma Mass Spectrometry, Anal. Chem., 73 (2001) 3006-3010. doi: 10.1021/ac010066s

John T. Rowan, R.S. Houk, Attenuation of Polyatomic Ion Interferences in Inductively Coupled Plasma Mass Spectrometry by Gas-Phase Collisions,  Appl. Spectrosc., 43/6 (1989) 899-1104. doi:  10.1366/0003702894204065

Related EVISA Resources:

Instrument database: Agilent Technologies Inc. - 8800 - Triple Quad ICP-MS
Brief summary: ICP-MS - A versatile detection system for speciation analysis
Brief summary: LC-ICP-MS - The most often used hyphenated system for speciation analysis

Related News:

March 10, 2012: Agilent Technologies publishes 2nd Edition of its Handbook of Hyphenated ICP-MS Applications

July 22, 2011: ICP-MS Analysis Suggests Metal-Binding Proteins Significantly More Abundant Than Thought

last time modified: July 29, 2012


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