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In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules Fahmida Jabeen, Muhammad Najam-ul-Haq, Matthias Rainer, Christian W. Huck, and Guenther Karl Bonn Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01573 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Analytical Chemistry

In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules Fahmida Jabeen1,2, Muhammad Najam-ul-Haq*1,2, Matthias Rainer2, Christian W. Huck2, Guenther K. Bonn2

1

: Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya

University, Multan 60800, Pakistan. 2

: Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain

80-82. Innsbruck 6020, Austria.

* Corresponding Author Prof. Dr. M. Najam-ul-Haq Institute of Chemical Sciences Bahauddin Zakariya University Multan 60800 Pakistan Tel.: +92 306 7552653 Email: [email protected]

Keywords:

In-tip

monolith,

Lanthanum

oxide,

Embedded

Phosphopeptides, Phospholipids, MALDI-MS.

ACS Paragon Plus Environment

poly(GMA/DVB)

tips,


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Abstract Polymeric monoliths fabricated in tips with embedded materials of choice are important in separation science. Polymeric backbone however interferes in the enrichment and thus affects efficiency. This work focuses on the in-tip fabrication of lanthanum oxide porous monolith and its application in the enrichment of phosphorylated peptides and lipids. Polycondensation reaction uses aqueous solution of LaCl3.7H2O with N-methyl formamide as porogen and propylene oxide as initiator. The ageing time of monolith and temperature condition for the reaction are optimized to attain porous monolithic tip. Comparison of (i) solid phase batch extraction using La2O3, (ii) La2O3 embedded in poly(GMA/DVB) tip and (iii) pure La2O3 monolithic tip shows improved enrichment efficiency in the case of pure La2O3 monolithic tip. Monolithic tip achieves selectivity of 1:4500 as compared to SPE (1:3500) and limit of detection down to 0.25 fmol. In-tip La2O3 monolith strategy has better batch to batch reproducibility, reduced time of enrichment and ease of operation in comparison to solid phase batch extraction. The developed strategy enriches phospho- content from biological samples like phosvitin and lipovitellin from egg yolk and phospholipids/phosphopeptides from human serum. The enriched phospho- moieties are analyzed by MALDI-MS except the phospholipids where LDI-MS is employed.


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Introduction Mass spectrometric detection and quantitation of phosphorylation is analytically challenging because of multiple reasons.1 Major reason is the transient nature of protein phosphorylation because of its reversibility that increases the difficulty of analysis2 and site annotation.3 Moreover the presence of interfering non-phosphopeptides suppresses the signal intensity of target

species.4

Hence

several

separation

techniques

are

developed

to

enrich

phosphopeptides/phosphoproteins by using phosphate group as interaction site. Reported strategies

include

precipitation5,

chemical

derivatization6,

hydrophilic

interaction

chromatography (HILIC)7 and ion exchange chromatography.8 Common techniques are immobilized metal ion affinity chromatography (IMAC; Fe3+ 9, Ti4+ 10, Zr4+ 11, Sm3+ 12 etc) and metal oxide affinity chromatography (MOAC; Fe3O413, TiO214, ZrO215, CeO216, La2O317 etc) which involve interaction of phosphate group with metals and metal oxides respectively. Different support materials (carbon based18, polymeric19, silica20, magnetic21) and formats (composites22, monoliths23) are introduced at micro/nano level for the purpose and still more combinations are being explored. Metal organic frameworks (MOFs) are the advancement towards enrichment of low-abundance phosphorylated peptides from complex mixtures. Incorporation of magnetic nanoparticles in MOFs offer good reusability and batch-to-batch repeatability.24,25 Metal-organic polymer hybrid (MOPH) with higher surface area and thermal stability are another separation media.26 MOFs with Zr-O clusters are used to incorporate UiO-66 and UiO-67 to exhibit molecular sieving effect with their use in phosphoproteome.27 MOF based monolithic capillary has been off-line coupled to matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for the analysis of phosphopeptides.28 Molecular imprinted polymers (MIPs) have also been



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the screening tools for specific phosphorylation sites.29 Preparation of MIPs is laborious and modifications in fabrication strategy have been formulated to access the potential of MIPs.30 Imprinted mesoporous materials show selectivity towards phosphopeptides, tolerance to interference, fast binding equilibrium, and larger binding capacity.31,32 One of the enrichment approaches for phosphopeptides is monolithic separation media, because of its advantages over the traditional stationary phases, i.e. flexible synthesis, adjustable porosity, fast mass transport, variety of surface chemistry, pH stability and biocompatibility.33 Poly(styrene-divinylbenzene) based monolithic columns are used for the separation of phosphorylated peptides by using higher and lower pH through THF, HFBA and TEA/HOAc as additives.34 The hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic layer is used for the separation of proteins in electrophoresis and pressurized planar electrochromatography.35 The modification of monoliths by embedding36,37 and coating38 of nanoparticles is a novel enrichment strategy having high surface-to-volume ratio. Synthesis of metal oxides by sol gel chemistry uses metal alkoxide as precursor.39 The alkoxides however require cautions as they are air sensitive and susceptible to hydrolysis.40 Development of monolithic structure using metal alkoxide involves fast reaction and quick gel formation which make it unsuitable for in-tip preparation. In-tip technology needs time between gelation and ageing so that polymerization mixture can be introduced into the tips. As second option, metal salts of zirconium and hafnium are experimented as precursors to develop respective metal-oxides.41 In this work, lanthanum oxide monolith is fabricated in tips for the faster extraction of phosphopeptides from biofluids. Comparison to conventional SPE batch extraction is carried out using β-casein digest. Studies like selectivity, sensitivity and reproducibility are performed to


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determine the enrichment efficiency of developed in tip monolith. Egg yolk digest is applied to enrich the phosphopeptides derived from phosvitin. Human serum is analyzed to assess the feasibility of successive elution to enrich phospholipids and phosphopeptides within one tip.



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Experimental Chemicals and Materials Lanthanum chloride heptahydrate (LaCl3.7H2O, 99.99%), n-methyl formamide, propylene oxide, glycidyl methacrylate (GMA, 97%), divinyl benzene (DVB, technical grade, 80%), decanol, azobisisobutyronitrile (AIBN), toluene, acetonitrile (ACN, reagent grade), methanol, β-casein (from bovine milk), (NH4HCO3), 2,5-Dihydroxybenzoic acid (2,5-DHB), dithiothreitol (DTT), iodoacetamide (IAA), trypsin, trifluoroacetic acid, ammonium hydroxide, chloroform, gold nanoparticles (4 nm) were purchased from Sigma Aldrich. Polypropylene tips (Eppendorf) were used to fabricate poly(GMA/DVB) embedded La2O3 tips and pure La2O3 monolithic tips. Commercial La2O3 (99.999% trace metals basis), TiO2 and ZrO2 for the embedding were purchased from Sigma Aldrich. TopTip (Glygen) and Phos-TiO2 (Titansphere, GL Sciences) were used for comparison.

Fabrication of In-Tip La2O3 Monolith Aqueous solution of LaCl3.7H2O (20 µL, 1 M) was prepared in deionized water. To the prepared solution, 150 µL of N-methyl formamide was added and sonicated for 5 minutes. Propylene oxide (166 µL) was added to induce the polycondensation reaction leading to polymerization. The mixture was aspirated into the tips to attain desired bed height. The tips were heated at 60 ⁰C for 3 hours. For temperature optimization, the tips were heated at 60, 80 and 100 ⁰C. The tips were allowed to polymerize for 3, 6, 9 and 12 hours to optimize time of ageing. The fabricated tips were finally washed with deionized water and stored at room temperature.


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Fabrication of In-Tip Poly(GMA/DVB) Embedded La2O3 Embedded tips were prepared according to the reported method.42 Polymerization mixture was prepared using 15 µL of glycidyl methacrylate (monomer), 15 µL of divinyl benzene (as crosslinker), 15 µL of decanol (porogen) and 2 mg azobisisobutyronitrile (initiator). Commercial La2O3 (1 mg) was added to the polymerization mixture and vortexed for 5 minutes. Five microliters of toluene were added and mixture was sonicated for 15 minutes at room temperature. The polymerization mixture was aspirated into tips and heated at 80 °C for 2 hours. The tips were then cooled to room temperature and washed with acetonitrile and methanol.

Sample Preparation The digestion of β-casein, egg yolk and human serum was carried out according to the reported protocol.43

Phosphopeptides Enrichment Protocol The monolithic tips were activated using ten cycles of aspiration and dispense with buffer (80% ACN in 0.1% (v/v) TFA). The tips were pre-equilibrated with 0.1% (v/v) TFA. Phosphopeptides were enriched from the sample digest (β-casein, egg yolk, serum) using 10 cycles for each sample. The tips were washed with 80% ACN in 0.2% TFA followed by DHB buffer (110 mg of DHB in 0.5% ACN) and deionized water. The elution was performed using 1.5% aqueous ammonium hydroxide solution for phosphopeptides and 1% ammonium hydroxide in chloroform for phospholipids [12]. The eluted fractions were subjected to MALDI-MS analysis. For comparison, embedded tips were used for enrichment from β-casein following similar protocol. For batch extraction, commercial lanthanum oxide was applied to β-casein and egg yolk. Similar buffers were used with incubation time of 45 minutes after sample loading.



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MS Analysis Enriched phospholipids from serum were mixed with commercial gold nanoparticles and analyzed by LDI-MS. Phosphopeptides were analyzed using dihydroxybenzoic acid (0.5 µL, 0.1% TFA: 50% acetonitrile (1:1) spiked with 1% phosphoric acid) as matrix. MS Analysis was carried out by using Ultraflex-II (Bruker Daltonics, Germany) MALDI-TOF/TOF-MS. Spectra were recorded in reflector mode using 337 nm UV laser at 50 Hz with 300 laser shots. The Flex Analysis version 3.3 and BioTools 3.0 software packages provided by the manufacturer were used for data processing. Data interpretation was performed by using online mascot server explored against SwissProt database with parameters: oxidation at methionine (M), phosphorylation at serine, threonine (ST) and tyrosine (Y), carbamidomethylation (C) and allowed missed cleavage 1. The identified phosphorylation sites are confirmed from Phosphosite plus and phospholipids by using Lipidmaps database (www.lipidmaps.org/).


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Results and Discussion Fabrication and Characterization During hydrolysis, lanthanum chloride heptaaqua is dissolved in deionized water to produce oxychloride. Lanthanum oxychloride further reacts with n-methyl formamide to produce intermediate precursor A. N-methyl formamide provides pores in the monolithic structure. With the precursor, addition of epoxide (propylene oxide) initiates condensation and polymerization resulting in the formation of gel. The polymerization mixture can thus be easily handled and moved into any desired mould. The reaction mechanism is presented in Figure S1. Ageing of monolith gives mechanical strength to the monolithic structure. Temperature and time are therefore optimized in the synthetic protocol to observe the effect of both parameters on porosity and stability of monolithic bed. For the temperature optimization, 60, 80 and 100 °C is applied keeping time of ageing constant. In case of 60 °C, 95% of tips are porous with stable bed height. For the second batch of 20 tips at 80 °C, only 40% of tips are porous where majority of the tips suffered breakage during aspiration, leading to unstable monolithic bed. At 100 °C, porosity is reduced and only 25% tips are in good shape. Representative graph for the temperature optimization (Figure S2a) reveals that the increase in temperature hardens the monolith packed in tips and once pressure is applied during aspiration/dispense cycle, either the monolithic bed breaks or detaches from side walls leading to poor reproducibility (Figure S2b-d). Porosity is also reduced which can be due to the reduced channels on increasing the temperature. In the next optimization step, tips are allowed to age for four different time durations (3, 6, 9 and 12 hours) while keeping the temperature constant. As time duration increases, the number of porous and stable tips decreases. According to the graph plotted for ageing time versus



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percentage of porous tips, 90%, 75%, 40% and 10% tips aged at 3, 6, 9 and 12 hours (Figure S3a). Longer ageing times cause shrinkage of monoliths which subsequently break the monolithic beds (Figure S3 b-e). Hence, optimized fabrication conditions for lanthanum oxide monolithic tips are 60 °C temperature and 3 hours ageing time for the adopted protocol. Scanning electron microscopic (SEM) images of poly(GMA/DVB) embedded La2O3 (Figure 1a) and La2O3 monolith (Figure 1b and c) show interconnected microstructures with prominent pores. EDX analysis of La2O3 monolith shows no impurity (Figure S4).

Comparison of Enrichment Strategies for Phosphorylated Biomolecules β-Casein digest is applied to La2O3 monolithic tips because of the known metal oxide affinity for phosphopeptides. La2O3 based materials show better performance as compared to IMAC based enrichment and other metal oxides like TiO2 and ZrO2 [17]. Conventional in tip strategy involves the embedding of affinity material in polymerization mixture. The embedded particles within monolith matrix show lesser adsorption capacity. In present study, poly(GMA/DVB) is used to develop monolith for the embedding of metal oxides like titania, zirconia and La2O3. Mass spectrum recorded for poly(GMA/DVB) without embedding shows number of nonphosphopeptides (Figure 2a). After embedding with TiO2 and ZrO2, number of phosphopeptides is enriched with the presence of non-phosphopeptides, which cannot be avoided because of the polymeric background (Figure 2b and c). Titania and zirconia show higher selectivity towards mono- and multi-phosphopeptides respectively. The enrichment efficiency is further enhanced on embedding La2O3 in poly(GMA/DVB) monolithic tip (Figure 2d). phosphopeptides

at

m/z

2061

(FQSEEQQQTEDELQDK,

Characteristic 48-63),

(KFQSEEQQQTEDELQDKIHPFΑ, 47-68), 2965 (RELEELNVPGEIVESLSSSEESITR, 40),

3054

(KKIEKFQSEEQQQTEDELQDKIHPFΑ,


43-68)

and

2556 163122

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(RELEELNVPGEIVESLSSSEESITRI, 16-41) are detected in the eluted fractions of embedded TiO2, ZrO2 and La2O3 tips. The data from fabricated La2O3 monolithic tips is compared with commercial La2O3 following the SPE protocol and the embedded La2O3 tips. Apart from phosphopeptides, both types of tips enrich two mono-phosphopeptides at m/z 1104 (KFQSEEQQQT, 47-56)

and 2432 (IEKFQSEEQQQTEDLQDK, 45-63) (Figure 3 b and c)

which are absent in the SPE strategy (Figure 3a). These phosphopeptides can be lost during washing step as material sites are more exposed in batch extraction or studding of affinity sites with abundant phosphopeptides during incubation. However, any conclusive reason is not found in the literature. The embedded tip suffers from interference due to polymeric backbone, resulting in non-specific bindings at m/z 1591.9 (VLPVPQKAVPYPQR, 185-198) and 1698 (SWMHQPHQPLPPTVMF, 158-171). Pure metal oxide monolith faces no such problem and results in better performance with no non-specific bindings and has increased number of target species. Two mono- at m/z 2779 (KIEKFQSEEQQQTEDELQDKIHPF, 44-67) and 3837 (RINKKIEKFQSEEQQQTEDELQDKIHPFΑQTQS, 40-72) and two multi-phosphopeptides at m/z 3605 (RELEELNVPGEIVESLSSSEESITRINKKI, 16-45)

and

3975

(RELEELNVPGEIVESLSSSEESITRINKKIEKF, 16-58) are detected in MS spectrum recorded for La2O3 monolithic tip (Figure 3c). A comparison is given for poly(GMA/DVB) embedded with TiO2, ZrO2, La2O3, and in-tip La2O3 in Table S1.

Sensitivity and Selectivity - Comparison of La2O3 SPE and Monolithic Tip Sensitivity or limit of detection is measured by using different molar dilutions (100, 75, 50, 25, 5, 1, 0.5, 0.25 femtomoles) of β-casein digest per mL of solution. These dilutions are applied to La2O3 as SPE material and as monolithic tip. The comparison shows higher sensitivity for in-tip La2O3 monolith as compared to SPE. The constant aspiration/dispense cycle provides efficient



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interactions of affinity sites with target species. The plot shows 0.25 fmol/mL detection limit for tips in comparison to 0.5 fmol/mL for SPE (Figure S5a). The selectivity study is carried out for La2O3 monolithic tip and SPE using spiked β-casein digest in BSA digest. Mixtures are prepared using various ratios of β-casein and BSA as 1:100, 1:500, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000 and 1:4500. After enrichment, graph is plotted between number of phosphopeptides detected and ratio of mixtures (Figure S5b). The comparison shows enhanced selectivity of tips (1:4500) as compared to SPE (1:3500). Selectivity is affected by the presence of background. BSA digest contains both hydrophilic and hydrophobic peptides which interact with hydroxyl group of metal oxide. More interaction time is available in SPE, therefore hydrophilic species bind to metal oxides and block the sites to interact with phosphopeptides.

Batch to Batch Reproducibility SPE is tedious and time consuming strategy whereas in-tip technology is quick and highly reproducible. Four batches of La2O3 monolithic tips are prepared and enrichment is performed using β-casein digest. Six characteristic peaks of reliable intensity are chosen and standard deviation is calculated for the respective m/z values (Table S2). The highest SD value is 0.75 for m/z 3122 and the lowest being 0.21 for m/z 2061. These two phosphopeptides are abundant and the difference in masses between theoretical (from database) and experimental is less.

Comparison with Commercial Materials Commercial materials like TopTip (TiO2/ZrO2 mixed, 1-10 µL tip with 4 mg of chromatographic media, Glygen) and Titansphere (1 mg TiO2 in 10 µL tip, GL Sciences) are selected for comparison with La2O3 monolithic tip. TopTip are the microspin columns packed with mixed


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metal oxides. β-casein digest is used as sample according to the provided protocol. Mass spectra from m/z 1500 Da to 3500 Da for the three materials show enrichment of phosphopeptides (Figure 3 a-c). With Titansphere, phosphopeptides at m/z 1473, 2061, 2556, 2965, 3054, 3122 and 3477 are detected (Figure 4a). TopTip enriches phosphopeptides at m/z 1473, 2061, 2476, 2779, 2965, 3122 and 3477 (Figure 4b). La2O3 monolithic tip enriches the highest number, i.e. 15 phosphopeptides (Figure 4c).

Application to Biological Fluids Egg yolk contains highly phosphorylated phosvitin which is derived from macromolecular lipophosphoprotein, vitellogenin. It serves as precursor for phosvitin (glycophosphoprotein) and lipovitellin. Phosvitin contains 55% of serine and majority is phosphorylated therefore one peptide may contain 6 to 10 phosphorylated peptides. The enzymatic specificity towards phosvitin is complicated due to its amino acid composition, degree of phosphorylation and glycosylation which lead to phosphopeptides that can be considered as non-enzymatic products. La2O3 monolithic tip is used to enrich phosphopeptides from egg yolk digest and for comparison enrichment is also performed by SPE strategy. The MS spectra show that number of phosphopeptides derived from phosvitin and lipovitellin are detected in both SPE and in-tip strategies (Figure 5 a-b). Phosphopeptides derived from phosvitin belong to domain I (11121145, AEFGTEPDAKTSSSSSSASSTATSSSSSSASSPN, PV 1-34) and III (1301-1322, SGHLEDDSSSSSSSSVLSKIWG, PV 190-211) while domain IV represents lipovitellin (IITEVNPESEEEDESSPYEDI, LP 1056-1076). The detected phosphopeptides after enrichment are listed in Table S3. Comparison of both strategies shows better performance of monolithic tip as compared to SPE. The difference can be explained in terms of retained multi-phosphopeptides on SPE material. La2O3 is also spotted after first elution using material enhanced LDI-MS



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method (MELDI®).44 In MS analysis, it is observed that most of phosphopeptides are detected in MELDI spectrum (Figure S6b) as compared to the first eluted fraction (Figure S6a). This indicates the need of multiple elutions to completely remove all the bound species from SPE material. The multiple elutions however prolong the enrichment procedure in SPE strategy. The in-tip monolith has inherent characteristic of multiple elutions achieved through the aspiration/dispense cycle. The monolithic tip is then used for human serum analysis. Digested serum (20 µL acidified with 0.1% TFA) is applied to La2O3 monolith tip by following optimized enrichment protocol. Successive elution is performed for phospholipids and phosphopeptides from serum. For phospholipids, LDI-MS analysis is carried out using gold nanoparticles as LDI matrix. Phospholipids like glycerophosphates, glycerophosphocholines, glycerophosphoethanolamines, glycerophosphoglycerols and phosphosphingolipids are detected (Figure 6). As phospholipids contain sphingoid bases and fatty acids as major components, they are also detected in mass range below 350 Da. In the successive second eluted fraction, serum phosphopeptides are detected and matched to search engine for the identification of phosphoprotein and annotated phosphorylation site. Mascot search against Swiss-Prot within given parameters identify 58 serum phosphoproteins which contain phosphorylation at serine, threonine and tyrosine (Table S4). The phosphorylation is manually confirmed using Phosphosite plus. Phosphorylation sites supported by more than 5 references are selected only. The eluted fractions can also be applied to LC-MS/MS for detailed identification of enriched content. Identification of phosphoproteins using the developed monolithic enrichment tips help in the biomarkers research. Modification site (phosphorylation site) on a specific protein is affected by non-synonymous Single Nucleotide Variations (nsSNVs). It is therefore necessary to explore the


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proteomic data at its genetic level. BioMuta® database is used to relate identified phosphoproteins with nsSNVs for different cancer types. The functional sites obtained from Swiss-Prot are mapped out with respect to nsSNVs (see Supporting Information). The data shows that phosphorylation is affected by nsSNVs. The online data include accession number, gene name, SNV position, polymorphism mutation prediction, disease ontology and source. Tabular data from database for the phosphoproteins identified during present research show variation of positions affected by specific cancer. Using this information, phosphorylation sites affected by nucleotide sequence can be related theoretically to a cancer. In mutations, the genetic sequence mutates the functional sites on an amino acid. Thus phosphoproteins which are assessed as potential biomarker for a cancer can be traced down to genetic mutations.



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Conclusion Separation science has produced range of enrichment approaches for the phospho- content. In-tip technology is flourishing because of its faster analysis and easier handling. Phosphoproteomics is a developing field and metal oxides are important enrichment materials in this regard. The lanthanum oxide monolith is fabricated in tips to enrich phosphopeptides from biological samples. Metal alkoxide is replaced with metal salts as precursor to synthesize the monolith. The temperature and ageing time are optimized to maintain porosity and stable monolithic bed. SEM images confirm the interconnected channels and porosity. Enrichment performance of La2O3 monolithic tip is compared with La2O3 as SPE material and embedded La2O3 tips with polymeric backbone. Experiments involving sensitivity, selectivity and application to real biofluids highlight the significance of tip technology over SPE. Conventional SPE can be replaced with more optimized monolithic tips of desired material for easier assessment of given sample in short duration.


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Acknowledgments This work is supported by the Higher Education Commission (HEC) of Pakistan and the University of Innsbruck, Nachwuchsförderung, Austria. Furthermore, the authors declare that they have no conflict of interest.

Supporting Information Available Additional information regarding the Mechanism of monolithic synthesis; Graphical representation of temperature and ageing time optimizations; Monolithic tips at 60 °C, 80 °C and 100 °C and Monolithic tips aged for 3 hours, 6 hours, 9 hours and 12 hours; Comparative MS analysis of egg yolk digest from phosvitin and lipovitellin; Tables regarding β-casein phosphopeptides to determine the batch to batch reproducibility, Phosphopeptides detected from egg yolk digest and Phosphoproteins identified from serum digest. This information is available free of charge via the Internet at http://pubs.acs.org. Ethical consent: Human serum samples were collected from the volunteers after taking their written consent and the approval by the ethical committee of the Institute.



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Figure Captions Figure 1: SEM micrographs of (a) La2O3 embedded poly(GMA/DVB) monolithic tip (b) La2O3 monolithic tip at zoom level of 5 µm and (c) La2O3 monolithic tip at zoom level of 1 µm.

Figure 2:

MS spectra of eluted fractions of β-casein digest after enrichment with (a)

poly(GMA/DVB)

without

embedding

(b)

poly(GMA/DVB)

embedded

TiO2

(c)

poly(GMA/DVB) embedded ZrO2 (d) poly(GMA/DVB) embedded La2O3 tip. Phosphopeptides are shown by symbol β. Common phosphopeptides (PPs) in three approaches are represented by for mono- and blue star

for multi-PPs. Red star

indicates mono- and

indicates PPs common in both types of tips. Filled

shows multi- PPs enriched by La2O3 monolithic tip. NP

represents non-phosphopeptides where “?” shows absence of PPs.

Figure 3: MS spectra of eluted fractions for β-casein digest after enrichment with (a) commercial La2O3 (b) La2O3 embedded poly(GMA/DVB) monolithic tip and (c) La2O3 monolithic tip. Phosphopeptides are shown by symbol β. Common phosphopeptides (PPs) in three approaches are represented by

for mono- and

PPs common in both types of tips. Filled blue star

for multi-PPs. Red star indicates mono- and

indicates the

shows multi PPs

enriched by La2O3 monolithic tip. NP represents non-phosphopeptides where “?” shows absence of PPs.

Figure 4: MS spectra of eluted fractions of β-casein digest after enrichment with (a) TopTip (Glygen) (b) Titansphere (GL Sciences) and (c) La2O3 monolithic tip. Phosphopeptides are shown by symbol β. Common phosphopeptides (PPs) in three approaches are represented by for mono- and

for multi-PPs. Red star

indicates PPs common in both types of tips. Filled


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blue star

indicates mono- and

shows multi- PPs enriched by La2O3 monolithic tip where “?”

shows absence of PPs.

Figure 5: Comparative MS analysis for egg yolk digest applied to (a) La2O3 as SPE material and (b) La2O3 monolithic tip. Phosphopeptides from phosvitin and lipovitellin are represented as and

respectively.

Figure 6: MS spectrum of the phospholipids detected from serum sample after enrichment using La2O3 monolithic tip.



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TOC Graphic


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Figure 1: SEM micrographs of (a) La2O3 embedded poly(GMA/DVB) monolithic tip (b) La2O3 monolithic tip at zoom level of 5 µm and (c) La2O3 monolithic tip at zoom level of 1 µm. 202x69mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2: MS spectra of eluted fractions of β-casein digest after enrichment with (a) poly(GMA/DVB) without embedding (b) poly(GMA/DVB) embedded TiO2 (c) poly(GMA/DVB) embedded ZrO2 (d) poly(GMA/DVB) embedded La2O3 tip. Phosphopeptides are shown by symbol β. Common phosphopeptides (PPs) in three approaches are represented by for mono- and for multi-PPs. Red star indicates PPs common in both types of tips. Filled blue star indicates mono- and shows multi- PPs enriched by La2O3 monolithic tip. NP represents non-phosphopeptides where “?” shows absence of PPs. 127x152mm (96 x 96 DPI)


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Figure 3: MS spectra of eluted fractions for β-casein digest after enrichment with (a) commercial La2O3 (b) La2O3 embedded poly(GMA/DVB) monolithic tip and (c) La2O3 monolithic tip. Phosphopeptides are shown by symbol β. Common phosphopeptides (PPs) in three approaches are represented by for mono- and for multi-PPs. Red star indicates the PPs common in both types of tips. Filled blue star indicates monoand shows multi PPs enriched by La2O3 monolithic tip. NP represents non-phosphopeptides where “?” shows absence of PPs. 120x152mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4: MS spectra of eluted fractions of β-casein digest after enrichment with (a) TopTip (Glygen) (b) Titansphere (GL Sciences) and (c) La2O3 monolithic tip. Phosphopeptides are shown by symbol β. Common phosphopeptides (PPs) in three approaches are represented by for mono- and for multi-PPs. Red star indicates PPs common in both types of tips. Filled blue star indicates mono- and shows multi- PPs enriched by La2O3 monolithic tip where “?” shows absence of PPs. 120x152mm (96 x 96 DPI)


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Figure 5: Comparative MS analysis for egg yolk digest applied to (a) La2O3 as SPE material and (b) La2O3 monolithic tip. Phosphopeptides from phosvitin and lipovitellin are represented as and respectively. 117x152mm (96 x 96 DPI)



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Figure 6: MS spectrum of the phospholipids detected from serum sample after enrichment using La2O3 monolithic tip. 276x150mm (96 x 96 DPI)


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