the benefits and limitations of reaction cell and …the beneﬁts and limitations of reaction cell...

Research Article

Received: 13 October 2014 Revised: 16 October 2014 Accepted: 16 October 2014 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2015, 29, 35–44

The benefits and limitations of reaction cell and sector fieldinductively coupled plasma mass spectrometry in the detectionand quantification of phosphopeptides

Evelyne Maes1,2,3*, Wilfried Brusten1, Filip Beutels1, Geert Baggerman1,2, Inge Mertens1,2,Dirk Valkenborg1,2, Bart Landuyt3, Liliane Schoofs3 and Kristof Tirez11Flemish Institute for Technological Research (VITO), Mol, Belgium2CFP-CeProMa, University of Antwerp, Antwerp, Belgium3KU Leuven, Research Group of Functional Genomics and Proteomics, Leuven, Belgium

RATIONALE: The phosphorylation of proteins is one of the most important post-translational modifications in nature.Knowledge of the quantity or degree of protein phosphorylation in biological samples is extremely important. Acombination of liquid chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) allows theabsolute and relative quantification of the phosphorus signal.METHODS: A comparison between dynamic reaction cell quadrupole ICP-MS (DRC-Q-ICP-MS) and high-resolutionsector field ICP-MS (SF-ICP-MS) in detecting signals of phosphorus-containing species using identical capillary LC(reversed-phase technology) and nebulizer settings was performed.RESULTS: A method to diminish the reversed-phase gradient-related signal instability in phosphorus detection with LC/ICP-MS applications was developed. Bis(4-nitrophenyl)phosphate (BNPP) was used as a standard to compare signal-to-noise ratios and limits of detection (LODs) between the two instrumental setups. The LOD reaches a value of 0.8 μg L�1 whenapplying the DRC technology in Q-ICP-MS and an LOD of 0.09 μg L�1 was found with the SF-ICP-MS setup. This BNPPstandard was further used to compare the absolute quantification possibilities of phosphopeptides in these two setups.CONCLUSIONS: This one-to-one comparison of two interference-reducing ICP-MS instruments demonstrates thatabsolute quantification of individual LC-separated phosphopeptides is possible. However, based on the LOD values,SF-ICP-MS has a higher sensitivity in detecting phosphorus signals and thus is preferred in phosphopeptide analysis.Copyright © 2014 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.7079

In a living organism, the post-translational modification ofproteins creates functional diversity, modulating andregulating the physiological activity of individual proteins.In this regard, reversible protein phosphorylation regulatedby kinases and phosphatases is one of most importantprotein modifications known.[1] In eukaryotic cells, theaddition of a phosphorus group takes place mainly at serine,threonine or tyrosine amino acid residues. This phosphorus-based modification creates changes in both proteinconformation and activity. The importance of the (de)phosphorylation event is reflected by the many processes itis involved in: regulation of metabolism, homeostasis,signal transduction, proliferation, differentiation and cellsurvival.[2,3] Perturbations in the (de)phosphorylation eventhave been linked to several pathologies including cancer.[4]

At present, over 100,000 sites for phosphorylation areknown in the human proteome.[5] However, determiningthe sites, the abundance of (de)phosphorylation events androle for each of these modifications in a complex biological

* Correspondence to: E.Maes, Flemish Institute for TechnologicalResearch (VITO), boeretang 200, 2400 Mol, Belgium.E-mail: [email protected]

Rapid Commun. Mass Spectrom. 2015, 29, 35–44

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sample is a huge and challenging task. The complexity ofthis task is not only due to phosphorylation dynamics, butalso because of the low relative abundance levels ofphosphorylation in proteins. To date, most of thephosphoprotein-based research is provided by molecularmass spectrometry, where an enriched phosphorylatedpeptide fraction is analyzed by matrix-assisted laserdesorption/ionization (MALDI) or electrospray ionization(ESI)-based mass spectrometry applications.[6] Althoughthese techniques are suited to identify these proteins andtheir phosphorylation sites, absolute quantification of thesesites necessitates synthetic phosphopeptide standards.[7]

These synthetic peptides for absolute quantification, stableisotope standard (SIS) peptides,[8] are, in combination withselective reaction monitoring (SRM),[9] a powerful tool thatallows the quantitative assessment of the levels of peptidesand their post-translational modifications. Production ofthese SIS peptides, on the other hand, is very expensiveand can be time-consuming.

Because absolute phosphopeptide quantification usingmolecular MS is thus not straightforward, other ‘elemental’approaches have been developed. Total reflection X-rayfluorescence (TXRF), for example, allows the determinationof the total amount of phosphorylation in biological

Copyright © 2014 John Wiley & Sons, Ltd.

E. Maes et al.

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samples.[10] For phosphoprotein/peptide quantification,inductively coupled plasma mass spectrometry (ICP-MS)coupled to liquid chromatography (LC) can be used as analternative approach.[11,12] In ICP-MS, absolute ‘phosphorus’quantification is possible because the obtained signal isproportional to the absolute amount of the monitoredelement. In protein research, this approach can thusdetermine naturally occurring elemental tags, likephosphorus or sulfur, in proteins.[13]

Even though ICP-MS is a very sensitive technique andallows signal detection of 9 orders of magnitude, thedetermination of phosphorus and sulfur is notstraightforward. Two of the major issues linked with P andS determination include: (1) their high ionization potentials(low sensitivity) and (2) the presence of polyatomicinterferences in their mass range. The monoisotopicphosphorus (m/z 31) signal, for example, is hindered by theexistence of polyatomic interferences, like argides, hydrides,carbides, nitrides and oxides, which are formed in the plasmaunder standard conditions. Also, for the determination ofsulfur in these plasma-based MS technologies, the signals ofthe two most abundant isotopes of sulphur, 32S and 34S, fallin a mass range with many interfering polyatomic species.[14]

To overcome these interference problems, moresophisticated ICP-MS techniques are required to allowaccurate phosphopeptide detection and quantification. In thisregard, several different instruments can be compared. Oneapproach which is used for phosphorus determination is aquadrupole ICP-MS, equipped with a collision or reactioncell. When the collision cell technology is applied, a collisiongas (e.g. helium) is introduced into the cell and allowsenhanced phosphorus detection, because the polyatomicinterferences (with larger atomic radii) are expected to losemore kinetic energy by collisions with the gas compared tothe ‘smaller’ analyte ions. Due to this loss in kinetic energy,an exclusion of interferences by kinetic energy discriminationbecomes possible.[15] On the other hand, in applicationswhere the dynamic reaction cell (DRC) is applied, the onlinereaction of phosphorus with e.g. oxygen (as reaction gas)enables the monitoring of PO+ (m/z 47) as analyte ion insteadof P+ (m/z 31).[16] In this way, the more abundantinterferences at m/z 31 are substituted by the less abundantinterferences at m/z 47. Another approach to obtaininterference-free phosphorus signals is by using a double-focusing sector-field ICP (SF-ICP) mass spectrometer. Theprinciple of this high-resolution instrument is to physicallyresolve analyte ions from interferences based on their smallmass differences. In SF-ICP-MS, ions are separated in amagnetic field with a high resolution capability up to10,000, in which case most interferences can be distinguishedfrom the analyte. For the determination of phosphorus andsulfur, a resolution of 4000 is sufficient to completely resolvethem from polyatomic interferences. Compared to quad-rupole instruments, higher ion transmission and lowervacuum conditions enables higher sensitivity.[17] However,this high-resolution instrument has two major disadvantages:(1) the instrument is 2 to 3 times more expensive incomparison with a quadrupole-based instrument and (2) ithas a reduced signal intensity at medium/high resolutioncompared to low-resolution measurements on the sameinstrument due to a reduction in transmission of theanalyte. Recently, a third ICP-MS instrument applicable in

wileyonlinelibrary.com/journal/rcm Copyright © 2014 John W

phosphorylation analysis, a triple quadrupole ICP massspectrometer, has been introduced. These instrumentsallows very efficient removal of polyatomic interferencesby applying tandem mass spectrometry (MS/MS).[18]

The possibilities of ICP-MS to accurately determinephosphopeptide concentrations in biological samples wereintroduced by Wind et al. in 2001.[19,20] To allow separationof different phosphopeptides, reversed-phase chromato-graphy was applied. In the context of ICP-MS, these organicsolvents can impose problems like signal enhancement orsuppression and carbon disposition at the cones, so specialprecautions were needed. As pioneers in the field ofphosphopeptide analysis with ICP-MS, Lehmann’s researchgroup downscaled the LC system to low μL min�1 combinedwith a low-flow total consumption nebulizer interface.[21] Asa result, only a limited amount of organic solvent isintroduced into the ICP instrument. However, even at theselow flow rates using capillary (4 μL min�1)[22] and nano(300 nL min�1)[23] HPLC setups, the organic mobile phaseshave a strong influence on the plasma ionization efficiency.To compensate for the gradient-dependent effects of theorganic mobile phases, different options including a constantor totally reversed post-column sheath-flow have beenproposed.[24] As a result, several research groups did applyLC/ICP-MS in the context of absolute phosphopeptidequantification and different limit of detection (LOD) valuesare provided for the different instrumental setups.[18]

Furthermore, new approaches, such as phosphorus-basedabsolutely quantified standard (PASTA) peptides,[25] havebeen developed. With this strategy, absolute quantificationnumbers are possible when the peak areas of peptides ofinterest are compared to the corresponding PASTA peptidesignal. The phosphorus present in these PASTA peptideshas been quantified with ICP-MS previously and comparisonbetween these two results thus in their molar ratio.[25]

To date, however, no one-to-one comparison betweendifferent phosphopeptide ICP-MS ‘detection’ options hasbeen provided yet in a setup where all the other settings(LC instrument and method, sheath flow construction, etc.)are similar. Therefore, the aim of this work is to compare thereaction cell quadrupole ICP-MS and the high-resolution sectorfield ICP-MS in detecting and quantifying phosphorus signalsusing identical capillary LC (reversed-phase technology) andnebulizer settings.

EXPERIMENTAL

Reagents

The mobile phases for the LC system were prepared withUHPLC gradient grade acetonitrile (ACN, Fisher Scientific,Erembodegem, Belgium) and uLC-MS grade formic acid(FA, Biosolve, Valkenswaard, The Netherlands). For alldilutions, dionized MilliQ water (18.2 MΩ) (Millipore,Billerica, MA, USA) was used.

Elemental standards for phosphorus (10,000 mg L�1),sulfur (10,000 mg L�1), indium (1000 mg L�1) in 2% HNO3,iron (10,000 mg L�1) in 5% HNO3 were purchased from SPEXCertiPrep (Metuchen, NJ, USA). Other ICP standards wereselenium (1000 mg L�1), arsenic (1000 mg L�1) andgermanium (1000 mg L�1) which were Certipur certifiedand obtained by Merck.

iley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2015, 29, 35–44

ICP-MS for the detection/quantification of phosphopeptides

Bis(4-nitrophenyl) phosphate (BNPP) standard (>99%) waspurchased by Sigma Aldrich (St. Louis, MO, USA). A stocksolution of 100 mg L�1 P dissolved in MilliQ water wasprepared and stored at 4 °C until further use.β-Casein (>99%) was purchased from Sigma Aldrich. The

lyophilized proteins were suspended in 100 mM triethylammonium bicarbonate buffer (Sigma Aldrich) (pH 8.0) toa final concentration of 3 mg mL�1 and heated to 90°C for20 min in order to denaturate and solubilize. Next, proteinswere digested using trypsin (Promega, Madison, WI, USA)in an enzyme/protein ratio of 1:25 during an overnightincubation at 37°C.

High-performance liquid chromatography (HPLC)

A Flexar system (PerkinElmer, Waltham, MA, USA) was usedas the chromatographic system, consisting of two Flexar FX-10UHPLC pumps, a Flexar solvent manager and a Flexar LCautosampler. For separation of phosphopeptides, a Zorbax SBRR C18 reversed-phase capillary column (150 mm×0.3 mm,5 μm particle size; Agilent) was used. A gradient with mobilephase A (5% ACN, 0,1% FA and 50 μg L�1 Ge) and mobilephase B (95% ACN, 0,1% FA and 50 μg L�1 Ge) was able toseparate the peptides using a flow rate of 350 μLmin�1. A flowsplitter was applied to allow a flow of 8 μL min�1 over thecolumn. The final LC settings are summarized in Table 1.A sheath-flow was constructed with a Harvard pump 11

Elite syringe pump (Harvard Apparatus, Les ulis Cedex,France) and an gastight glass syringe (2.5 mL; Hamilton).The sheath-flow solution contained 40% ACN, sulfur(20 mg L�1), arsenic (0.1 mg L�1) and selenium (0.1 mg L�1).

Inductively coupled plasma mass spectrometry

In this study, two different types of ICP-MS instrument wereused. The first instrument is a quadrupole-based ICP massspectrometer equipped with a collision/reaction cell (Nexion300S; Perkin Elmer, Waltham, MA, USA). The second is adouble-focusing magnetic sector field instrument (Element2; Thermo Scientific, Waltham, MA, USA). It should be notedthat both instruments are normally used for speciationstudies and not for peptide analysis. Therefore, a DS-5microflow total consumption nebulizer (S-prep GmbH,Uberlingen, Germany) and low-flow spray chamber were

Table 1. Experimental settings for the UHPLC system

Parameter Setting

Column Zorbax SBRRC18, 150mm×0.3mm;5 μm particles

Flow rate 350 μl min–1; flow splitter beforecolumn8 μL min�1 over column

Loop size 10 μL; fixed loopInjection volume 10 μLTotal LC run time 25 minGradient0 min -> 0.2 min 5% B0.2 min -> 6.7 min 5% B -> 40% B6.7 min -> 8.2 min 40% B -> 75% B8.2 min -> 8.5 min 95% B8.5 min -> 25 min 100% A

Rapid Commun. Mass Spectrom. 2015, 29, 35–44 Copyright © 2014 Jo

used to compensate for the low LC flow rate of ca. 8 μLmin�1.In the construction of the post-column sheath-flow, a T-pieceallowed mixing of the outlet capillary (50 μm I.D., 360 μmO.D.) of the capLC column with the sheath-flow solutionbefore introduction into the ICP-MS setup. The finalexperimental parameters of the Q-ICP-MS and SF-ICP-MSsetups are described in Tables 2 and 3.

Quadrupole DRC-ICP-MS: Tuning

In order to obtain the best conditions, the quadrupole ICP-MSinstrument was tuned daily. First, a standard ICP tuning with1 μg L�1 In in 2% HNO3 was performed to obtain a goodsensitivity (>60,000 counts per cps; cyclone spray chamber)and to find the optimal torch alignment and lens voltages.When optimal settings are achieved, the total consumptionnebulizer is installed. Next, before the coupling of the capLCto the nebulizer, a continuous flow of 10 μL min�1 wasdirectly infused with a syringe pump. Further optimizationof the gas flows (nebulizer gas flow; auxiliary gas flow) wasperformed with a 100 μg L�1 phosphorus solution containing40% ACN. The optimal conditions were found when a highstability and sensitivity of the PO47+ signal was achieved. Thepresence of organic solvents in these tuning solutions allowedus to find the optimal conditions in a situation that mimics thetotal reversed-phase gradient to the most possible extent.

For the implementation of the dynamic reaction cell (DRC)technology in our quadrupole ICP-MS instrument, reactioncell gas flows were optimized. To determine the optimal O2

cell gas flow in the DRC module, three different solutionswere directly infused with a syringe pump at a flow rate of10 μL min�1 to mimic the LC conditions: (1) a 40% ACNblank solution and (2) a 100 μg L�1 P standard in 40% ACNsolution. The signal intensities of both the blank and thephosphorus-containing sample are evaluated. The O2 cellgas flow rate with the highest cps signal (after backgroundsubtraction of blank) was 0.4 mL min�1. Therefore, a cellgas flow of 0.4 mL min�1 was selected for all furthermeasurements. With these adjustments, the signalbackground at 31P (m/z 31) (ca. 500,000 cps) in standard modecan be reduced to a background around 2000 cps at 31P16O(m/z 47) in DRC mode.

Table 2. Experimental settings for Q-ICP-MS

Parameter Setting

Type of instrument Nexion 300S, Perkin ElmerNebulizer gas flow 1.12 Lmin�1

Oxygen gas flow 0 Lmin�1

Auxillary gas flow 1.2 Lmin�1

Plasma gas flow 18 Lmin�1

ICP RF power 1500 WAnalog stage Voltage �1600Pulse stage voltage 1700Discriminator threshold 15Deflector voltage �8RPa 0,02RPq 0,45Cell gas flow 0.4 mL min�1

Sampling cone PtSkimmer cone Pt

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Table 3. Experimental settings for SF-ICP-MS

Parameter Setting

Type of intsrument Element II, Thermo ScientificNebulizer gas flow 1.16 Lmin�1

Oxygen gas flow 0 Lmin�1

Auxillary gas flow 0.89 Lmin�1

Plasma cool gas flow 16 Lmin�1

ICP RF power 1250 WResolution MediumSampling cone PtSkimmer cone Pt

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Sector-field ICP-MS: Tuning

The optimal settings in the sector-field ICP-MS setup wereachieved by tuning the instrument first with an aqueoussolution containing 1 μg L�1 In and μg L�1 Fe in 2% HNO3

by means of a cyclonic spray chamber. This tuning wasperformed in low resolution (m/Δm=400) and allows to findthe optimal torch alignment and lens voltages. Next,determination of mass offset was performed with an aqueoussolution (1% HNO3) containing a 100 μg L�1 concentration ofthe following elements: phosphorus, sulfur, arsenic, selenium,germanium, and indium. To optimize the gas flows, theconstruction with the DS-5 nebulizer and syringe pumpallowed to directly infuse a 40% ACN solution containing100 μg L�1 P, S, As and Ge at a flow rate of 10 μL min�1.The optimal stability and sensitivity of the system wereobtained by monitoring the 74Ge signal in low resolutionand the 31P, 32S and 75As signals in medium resolution (m/Δm=4000). The presence of organic solvents in these lattersolutions allows the optimal conditions to be found in asituation that mimics the total reversed-phase gradient tothe greatest possible extent.

RESULTS AND DISCUSSION

Although the potential of ICP-MS in the absolutequantification of phosphopeptides was demonstrated a decadeago, the integration of this elemental technique inphosphoproteomics studies is still very limited. Therefore, inthis work, we discuss the ICP-MS technology in its efficiencyof phosphorus detection with the emphasis on their potentialsand limitations in phosphopeptide characterization. For thispurpose, we compare the two most commonly used ICP-MSinstruments in their efficiency of phosphopeptide detectionwhen identical capLC and sample introduction conditions areprovided. These two instruments include a quadrupole ICP-MS instrument (equipped with collision/reaction cell) and adouble-focusing sector-field ICP-MS instrument (operated atmedium resolution), with the latter one being 2 to 3 times asexpensive as the first device. Both instruments are comparedin terms of limit of detection (LOD) when they are hyphenatedwith the same UHPLC instrument, and when the samecapillary LC conditions and nebulization system are applied.To compensate for the organic solvent gradient-related

effects, a constant post-column sheath-flow setup is constructedas described in the literature.[26] Although we are aware of thefact that this constant post-column sheath-flow addition using


a syringe pump is less robust and less compensatory comparedto the totally reversed post-column sheath-flow constructiondescribed by Profrock and Prange,[24] it still provides adequateresults in the elution window of most of the trypticphosphopeptides. Additionally, it is a simple adjustment tomake the ICP-MS instrument compatible with a capLCworkflow for bioorganic analytes. The experimental capLC/ICP-MS setup used for phosphopeptide detection in this studyis visualized in Fig. 1.

Compensating for reversed-phase gradient-related effects

The online coupling of an LC gradient with carbon-containing compounds (e.g. organic solvents) to an ICP-MSinstrument will change a number of ICP-related processesalong with this gradient. These parameters include theplasma temperature, the ionization process in the plasmaand carbon deposition at the cones.[27] Also, a signalenhancement or suppression during this gradient is animportant factor which should be considered. All thesefluctuations in ICP-MS sensitivity during gradient elutionwill complicate accurate and absolute quantitative analysis.

To study the effect of the sheath-flow, we infused increasingconcentrations (5% increase for each solution) of organicsolvent at a flow rate of 10 μL min�1. For each concentrationof ACN, two solutions were prepared: (1) a blank solution ofACN and (2) the same blank solution with 20 mg L�1 S and100 μg L�1 P, As and Se added. Both solutions are preparedin an ACN concentration sequence starting with 10% ACNuntil 50% ACN. These solvent concentrations were chosenas they represent the concentrations of organic modifierspresent in the elution region of phosphopeptides (i.e. 10%–50% ACN). All these measurements are performed using aquadrupole instrument in DRC mode. Because a sheath-flowis created with 40% of ACN, we used this concentration as arelative intensity of 100%. The blank-subtracted relativeexpressed signals of phosphorus, sulfur, arsenic and seleniumare summarized in Fig. 2.

Although the same amounts of phosphorus, sulfur,selenium, and arsenic are provided in every solution, majordifferences in signal intensity are visible in the organic solventconcentration range where peptides elute. For example, a 3-fold increase in signal intensity is observed between 10%and 50% ACN for the studied elements, even at capillary flowrates (10 μL min�1). This increase in signal intensity inplasma-based techniques is well known and can be relatedto charge transfer reactions from ionized carbon to themonitored elements.[28] Furthermore, in a complete LCgradient ranging from 9.5 to 90.5% ACN, a factor of 4 insignal increment is observed for the measured elements inDRC-Q-ICP-MS (e.g. 34S16O: Smin = 14,980 cps; Smax = 59,236cps) as well as for SF-ICP-MS (e.g. 32S: Smin = 186,886 cps;Smax = 818,819 cps).

To minimize this gradient-dependent influence in thepeptide elution region (10%-50% ACN), two concomitantstrategies are proposed for LC-ICP-MS applications. First, aconstant post-column sheath-flow of 40% ACN (4 μL min�1)was added to the LC column eluent (8 μL min�1) beforeintroduction and nebulization of the solution in the ICPinstrument. With the implementation of the constant sheath-flow construction, a decrease to a 1.5-fold signal enhancementalong the peptide elution window in the LC chromatogram is


Figure 1. Overview of the capLC/ICP-MS experimental setup with sheath-flow construction.

Figure 2. Representation of the influence of the acetonitrileconcentration on the relative signal intensity of severaloxygenated elements: 31P16O, 34S16O, 75As16O, 81Se16O.


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achieved(e.g. DRC-Q-ICP-MS (e.g. 34S16O: Smin = 15,160 cps;Smax = 26,293 cps). This is the result of a continuouslybuffered LC eluent along the gradient via the sheath-flow,so that solvent concentration ranges in this peptide elutionregion are between 20 and 45%. Although we know that ahigher sheath-flow versus column ratio would benefit thesignal stability,[26] these low flow rates are not technicallyachievable with our capLC system. Additionally, highpressures are present in this sheath-flow construction using asyringe pump, with higher sheath-flow rates resulting inpossible leaks.Secondly, an elemental correction factor was applied to

reduce this variation in signal intensity across the gradient.To this end, we tested whether ratios of phosphorus todifferent elements (S, Se and As), which all have similarplasma behavior characteristics compared to phosphorus,could result in an enhanced stability over the peptide


gradient. The signal ratio of phosphorus to these elements isvisualized in Fig. 3. In the DRC-Q-ICP-MS setup, the31P16O/34S16O ratio is more stable compared to other signalsand a relative standard deviation (RSD) value of 8% in thispeptide elution window is achieved. Similar observationswere made when the element ratios are measured with SF-ICP-MS. Here, the 31P/32S ratio only had an RSD of 5% overthe elution window. The detection of sulfur, however, underconditions where organic matrices are present, is stillchallenging due to the interfering ion species present. Theinterferences which pop up in DRC mode with the use oforganic solvents are identified as carbon-containing ions:36Ar12C+ (47.949 u) interfering with m/z 48 and 38Ar12C+

(49.9579 u) and 36Ar14N+ (49.9661u) as interferences at massm/z 50.[14] Although the most abundant isotope of sulfur ispreferred for measurement (S32), we opt to use the 34SOisotope signal in the DRC mode, because, under ouroptimized conditions, less background is present at thissulfur isotope.

Although the use of this P/S ratio was also recommendedbyWind and colleagues more than a decade ago,[19,29] we willnot use the sulfur present in peptides (e.g. in methionine) asan internal standard to quantify both the phospho anddephospho forms of peptides and proteins. Rather, we haveintroduced a major amount of the element (S concentrationis 20 mg L�1) in the sheath-flow solution. The concentrationof S from the sheath-flow (80 ng Smin�1) is high whenconsidering the amount of S from samples, e.g. 100 pmol ofβ-casein contains a total of 25 ng S in its digest, resulting fromthe presence of sulfur-containing amino acids (7 methionineand 1 cysteine). In addition, we should point out that theamount of S per minute from the sheath-flow is constant,while the amount of S per minute from the β-casein digestdepends on the chromatographic characteristics of thepeptides. The total amount of S from the digest will thusnot elute at one time point. In addition, by implementing asheath-flow with a high amount of sulfur, the interferencesignals at the SO+ or S masses, arising from the presence oforganic solvents,[14] are also negligible, as correct isotope


Figure 3. Visualization of the corrected signal intensities in the peptide elution window. A comparison between 31P16O/34S16O;31P16O/75As16O; 31P16O/81Se16O and 31P/32S indicates that correcting the signal by a ratio of phosphorus-to-sulfur achieves themost stable signal across the peptide elution window.

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ratios (32S16O/33S16O=125.9935; 33S16O/34S16O: 0.134 and32S/33S = 126.29; 33S/34S: 0.161; theoretical values: 126.69and 0.178) are observed.With the implementation of both the sheath-flow and the

P/S correction, a more stable signal/sensitivity is attainableand a 3-fold sensitivity difference due to gradient-dependency of the signal intensity can be reduced to a RSDof 8% on the P/S signal in the peptide elution window. Thisincreasing signal stability allows amore accurate quantificationof phosphorus-containing species eluting across the organicsolvent gradient.

Bis(4-nitrophenyl) phosphate standard to determine the LOD

The absolute and accurate quantification of phosphopeptidesrequires, besides signal stability achieved by the sheath-flowand P/S correction, also a phosphorus-containing standard.In most ICP-MS setups, the search for a reliable standard isless complex compared to molecular MS because, in


elemental MS, a species and structure independency ispresent. Therefore, a species-independent quantification ofphosphopeptides is possible, which allows to circumventthe need for synthetically produced protein standards thatare required to obtain absolute quantitation information inmolecular MS applications. To this end, Navaza and co-workers compared different phosphorus-containingcompounds and recommended the use of bis(4-nitrophenyl)-phosphate (BNPP) because this molecule is retained on thecolumn and elutes closely to, but not in, the elution windowof peptides.[26] We will therefore also implement BNPP asreference material to allow conversion of the ICP-MS signalinto absolute numbers of peptide phosphorylation.

In a first experiment, the linearity of BNPP during areversed-phase capLC gradient elution was tested in boththe DRC-ICP-MS and SF-ICP-MS setups. In this comparison,different concentrations of BNPP (100 μg L�1, 10 μg L�1,5 μg L�1, 1 μg L�1) were quantified after elution during a25 min LC gradient using the area under the curve (AUC).



In these settings, the LC gradient with a flow rate of8 μL min�1 was compensated with a constant sheath-flowof 4 μL min�1. With both instrumental setups (DRC-Q-ICP-MS vs SF-ICP-MS), a calibration curve with a linearity ofR2 = 0.999 was obtained when the AUC values of the P/Scorrected signals were integrated (Supplementary Fig. S1,measured with the DCR-Q-ICP-MS (left panel) and SF-ICP-MS (right panel) instrument, see Supporting Information).To further compare these two instrument setups,

differences in peak area, signal-to-noise ratio, limit ofdetection (LOD) and quantification limit (LOQ) forphosphorus-containing species were calculated based on theBNPP standard. An overview of this comparison is providedin Table 4.When the peak area of a 100 μg L�1 BNPP injected solution

is calculated in a P/S corrected chromatogram (Fig. 4), a20-fold difference is observed between DRC-Q-ICP-MS(33) and SF-ICP-MS (750). Comparing signal-to-noise ratios,with the noise calculated as the standard deviation of theP/S-corrected signal of a 1 min background noise window,however, downscaled the factor to a 2-fold difference(Fig. 4). These results underline the benefits of using thedynamic reaction cell to lower background noise bymonitoring SO+ as analyte ion instead of S+.Furthermore, the detection limits, calculated by three

times the standard deviation of the P/S-corrected signal ofa 1 min background noise window, were as low as 0.8 μg L�1

(or 25 fmol P absolute) for the DRC setup (31PO/34SO) and

Table 4. Overview of comparison between DRC-Q-ICP-MS and SF-ICP-MS

LOD LOQ Linear range

DRC-ICP-MS 0.8 μg L�1 1.6 μg L�1 R2 = 0.999SF-ICP-MS 0.09 μg L�1 0.18 μg L�1 R2 = 0.999

Figure 4. The phosphorus-to-sulfur corrected LC signal infunction of the relative retention time of a 100 μg L�1 BNPPpeak measured by both DRC-Q-ICP-MS and SF-ICP-MS.Comparison of the signal-to-noise (= standard deviation of a1 min noise window) ratio demonstrates that a difference ofa factor 2 in peak detection is observed.


41

0.09 μg L�1 (or 2.9 fmol P absolute) when applying SF-ICP-MS (31P/32S), indicating a 10-fold difference in sensitivitybetween the two instruments. The LOQ values, calculatedas six times the standard deviation of the P/S-correctedsignal in a 1 min background noise window, aresummarized in Table 4. While in several studies differentdetection limits for phosphorus detection using DRC aredescribed [Bandura et al.[16] (LOD: 0.2 μg L�1), Ciavardelliet al.[30] (LOD: 0.4 μg L�1), Smith et al.[31] (LOD: 2.2 pmol)],none of these LOD observations were made using areversed-phase capLC gradient. Although rigorouscomparison of these data is not feasible, the sensitivitiesobserved in the reported data are quite comparable.Regarding the comparison of LOD values of phosphorusdetection using SF-ICP-MS, two other publications whichapply a reversed-phase capLC gradient to detectphosphopeptides are described. Our LOD results (2.9 fmol)(with sheath-flow and P/S correction) are comparable withthe described LODs of 100 fmol (only P/S correction)[19]

and 50 fmol (only gradient-based correction factor).[25]

Although no precise numeric comparison is possiblebetween DRC-ICP-MS and SF-ICP-MS due to differences inion optics and vacuum settings, some interesting trends canbe demonstrated. For example; switching from the low-resolution mode (m/Δm=400) to medium-resolution mode(m/Δm=4000) in SF-ICP-MS is accompanied by a 10-folddecrease in sensitivity, which is of the same order as expectedfor the quadrupole ICP-MS setup. However, because of theinterferences at the m/z 31 mass, phosphorus signals can onlybe accurately detected when using a collision or reaction cell.When applying oxygen as reaction gas, however, lower-than-expected signal intensities are observed, indicating that asubstantial loss in absolute sensitivity, P+ versus PO+, is alsoobserved when measuring in oxide mode.

Absolute quantification of phosphopeptides

In the literature, the absolute quantification of phosp-hopeptides is demonstrated using bovine β-casein.[18,19,29,32]

This model protein is known to yield two phosphopeptides(one monophosphorylated and one tetraphosphorylatedpeptide) after tryptic digestion. In this study, the modelprotein was used for the comparison of the phosphopeptidequantification capabilities of both the DRC-Q-ICP-MS as wellas the SF-ICP-MS setup. To allow the reliable quantification ofthese two phosphopeptides a clear separation of the twopeptides in an LC gradient is required. On top of that, in eachsample, a phosphorus standard – BNPP in this case – waspresent to permit compound-independent quantification ofbiomolecules.

To test the detection and quantification capabilities of bothICP-MS instruments, four samples each containing the sameamount of BNPP (100 μg L�1) and a decreasing amount ofβ-casein digest (injected quantity on the column: 2.5 μg;1.25 μg; 0.25 μg and 0.05 μg, which represents 100, 50, 10and 2 pmol, respectively) were prepared and analyzed bycapLC/ICP-MS, with the same LC and nebulizationconditions for both ICP-MS instruments. Figure 5(A) displaysthe PO47/SO50-corrected LC/ICP-MS chromatogram of atryptic digest of β-casein (1.25 μg of digest injected onto thecolumn). The first small peak is the peak of themonophosphorylated peptide, followed by the elution of the


Figure 5. The separation and quantification of two phosphopeptides of β-casein. (A) The chromatogram of a tryptic β-caseindigest (50 pmol absolute) loaded onto the column. (B) Demonstration that measuring four times the same internal standardresults in an RSD of 0.6%. The calibration curves of a monophosphorylated β-casein peptide (C) and a tetraphosphorylatedpeptide (D) are provided.

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second phosphopeptide at 14 min. As expected, a third peak,the BNPP peak, elutes close to the peptide elution region,ensuring the control on similar sensitivity conditions.[26]
Figures 5(C) and 5(D) demonstrate that increasing amountsof both phosphopeptides could be quantified (AUCs) withgood linearity’s (R2 of 0.99) in the DRC setup (lowest pointclose to LOD). Similar observations are seen with SF-ICP-MS. Furthermore, repetitive measurements of the sameamount of BNPP in these LC runs do show that low RSDvalues (0.6 %, n= 4) of the P/S-corrected peak areas couldbe obtained (Fig. 5(B)). This observation ensures that areproducible absolute quantification of phosphopeptidepeaks is possible and that minimal signal drift is present.The absolute quantification of both phosphopeptides is

possible when the PO/SO-corrected BNPP calibrationcurve is implemented in the obtained results. For


example, when 1.25 μg of the tryptic β-casein digest(50 pmol absolute) is loaded onto the column (representedin the chromatogram of Fig. 5), absolute amounts of14.49 pmol P and 47.69 pmol P for the first and secondeluted phosphopeptide are found, respectively. A 5-folddilution of the β-casein sample resulted in 2.7 pmol Pand 10.7 pmol P for the two phosphopeptides,respectively. This 1:4 phosphorylation stoichiometry isin agreement with the observation that in the digestedβ-casein sample, a mono- and tetra-phosphorylatedpeptide is present.

In analogy with the previously mentioned data in theliterature, our obtained results demonstrate that absolutequantification of phosphopeptides using the capLC/ICP-MS setup with sheath-flow and P/S correction is possible.The S signal, used to compensate for changes in sensitivity



along the elution window, is not significantly hampered bypossible signals from S-containing peptides due to the highconcentration of S in the sheath-flow.Currently, the capability of ICP-MS in the context of

absolute quantification of individual phosphopeptides islimited to non-complex samples. The main reason for this isthe fact that ICP-MS is an elemental detector, and thus aseparation up to the level of individual phosphopeptides isrequired, because all structural and identification informationis thus lost. In this regard, an extended fractionation might bepossible to obtain individual peptides; however, the questionremains whether ICP-MS technologies will ever be able todetect these ultra-low peptide concentrations.

ICP-MS as a relative screening tool

Besides the absolute quantification of individual phospho-peptides, ICP-MS can also be used as a screening tool todetect changes in phosphorylation across different samples.In this regard, LC/ICP-MS can be applied to check thepresence of phosphopeptides or proteins in samples or toobserve relative differences in overall phosphorylation peakpatterns of different samples.[33]

In the case of relative screening, no accurate quantificationis required, and thus no P/S correction is strictly necessary.A comparison between the signal-to-noise ratios (in fact,the signal to standard deviation of a 30 s noise window closeto peak) of both DRC-Q-ICP-MS and SF-ICP-MS of a β-caseindigest spiked with BNPP demonstrates that, in this case, theuse of SF-ICP-MS is beneficial to observe relative differencesin overall phosphorylation peaks whenever low noise levelscan be achieved (Fig. 6). As the distance between thedifferent chromatographic peaks is small, a 30 s time framewas preferred instead of the 60 s window used previously.The most important aspect in relative quantification is toobtain reproducible chromatograms during LC separation.In our setup, both instruments demonstrate that a goodreproducibility (of the BNPP peak area) can be obtained(DRC-Q-ICP-MS: RSD= 8.9%; SF-ICP-MS: RSD= 3.3%).However, with P/S correction, an even better reproducibility

Figure 6. A comparison between the signal intensities andsignal-to-noise of DRC-Q-ICP-MS and SF-ICP-MS in thedetection of phosphorus signals (β-casein digest and BNPPstandard).


(RSD=0.6%) can be achieved. This high reproducibility booststhe potential of the technique as a screening tool, as introducedby the Caruso lab, in the observation of relative differences inoverall phosphorylation in different samples.[33]

CONCLUSIONS

In this work, a one-to-one comparison between DRC-Q-ICP-MS and SF-ICP-MS instruments in the detection andquantification of phosphorus-containing species has beenconducted for the first time. Although a profound effect ispresent when organic solvents are introduced in ICP-MSinstruments, and several methods that allow to circumpassthese limitations partially have been described, we havedemonstrated that the implementation of (1) a sheath-flowconstruction that compensates the organic solvent gradientand (2) a P/S correction factor that can minimize the gradient-related effects to the best possible extent, is beneficial.Furthermore, our results do indicate that absolute quantificationof individual phosphopeptides in non-complex samples ispossible with BNPP as phosphorus standard. Obtainingabsolute numbers of phosphorylation of individual peptides incomplex biological samples (e.g. human blood samples), onthe other hand, is currently not possible using currentLC/ICP-MS configurations, because extended fractionationstepsmight also result in non-detectable ICP-MS concentrations.

AcknowledgementsThe authors are thankful to Dr. Jorge Ruiz Encinar forassistance with the experimental setup. This work wassupported by an Emmanuel Van der Schueren grant ofVlaamse Liga tegen kanker (VLK) and the Flemish Institutefor Technological Research (VITO).

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