MS Analysis of

Chip-Based Enrichment and NanoLC-MS/MS Analysis of Phosphopeptides from Whole Lysates Shabaz Mohammed,† Karsten Kraiczek,‡ Martijn W. H. Pinkse,†,§ Simone Lemeer,† Joris J. Benschop,†,⊥ and Albert J. R. Heck*,† Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, and Agilent Technologies R&D and Marketing GmbH & Company KG, Hewlett-Packard-Strasse 8, 76337 Waldbronn, Germany Received October 3, 2007

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated ‘nanoflow’ analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO2 particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates. Keywords: Automated • Phosphopeptide Enrichment • TiO2 • Chip

Introduction Post-translational modification (PTM) of proteins is nature’s way to (transiently) modify protein function and/or activity or to tag proteins for specific subcellular routing.1 Protein phosphorylation may be the most widespread and thus possibly most important PTM.2–4 Therefore, it should be no surprise that significant efforts have been made to develop methods to enable the study of cellular phosphorylation events. However, progress in attaining site localization and expression level in context of cell/protein state has been slow primarily due to the intrinsic nature of the phosphorylated residue. For instance, phosphorylated peptides are present at substoichiometric levels, undergo facile fragmentation inside a mass spectrometer to create potentially difficult to interpret mass spectra, and may provide poorer signal responses than their unphosphorylated counterparts.5 A plethora of techniques have been developed addressing several of these issues, primarily based on the specific enrichment of the phosphopeptides of interest. Phosphopeptide enrichment strategies include the use of phosphorylated residue specific antibodies,6–8 immobilized * To whom correspondence should be addressed. E-mail: [email protected]. † Utrecht University. ‡ Agilent Technologies R&D and Marketing GmbH & Company KG. § Present address: Analytical Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. ⊥ Present address: Department for Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. 10.1021/pr700635a CCC: $40.75

 2008 American Chemical Society

metal cation affinity chromatography (IMAC),9–11 with its more recent variants based on metal oxides of zirconium,12 aluminum,13 and titanium,14–17 strong cation exchange (SCX)18 or strong anion chromatography exchange (SAX).19,20 Comprehensive reviews describing these enrichment techniques are available.21,22 Moreover, a number of mass spectrometric methods have been developed targeted at the specific analysis of phosphorylated peptides including precursor ion scanning using diagnostic phosphorylated peptide fragments,23,24 neutral loss scanning exploiting similarly diagnostic fragmentation pathways,25 multiple reaction monitoring (MRM),26 multiple stages of fragmentation,27,28 and, most recently, electron transfer dissociation.29,30 Despite all these considerable efforts, it remains a challenge to perform comprehensive identification of protein phosphorylation sites.31,32 Microfluidic HPLC-Chip/MS technology introduced over the past decade provide novel platforms for peptide separation and is establishing itself as a robust, reliable alternative to conventional nanocolumn LC-MS systems.33 Potential benefits are provided by the integration of the enrichment column, analytical columns, connecting capillaries, and nanospray emitter directly onto the microfluidic HPLC-Chip device, which was demonstrated to lead to improved sensitivity and enhanced chromatographic performance leading to superior peptide and protein identification.34,35 Such chips have been shown to be more than acceptable replacements for traditional nanoLC setups for a number of applications.36–39 Utilizing these Journal of Proteome Research 2008, 7, 1565–1571 1565 Published on Web 02/29/2008

research articles advances in microfluidic HPLC-Chip/MS technologies, we report here on the optimization of a chip design that combines the power of TiO2-based phosphopeptide enrichment with the microfluidic HPLC-chip technology.

Materials and Methods Sequencing grade trypsin was purchased from Roche Diagnostics (Ingelheim, Germany). Bovine serum albumin, alpha casein, beta casein, and hemoglobin were from Sigma-Aldrich (Zwijndrecht, The Netherlands). Ammonium bicarbonate, sodium phosphate, potassium fluoride, potassium chloride, sodium orthovanadate, dithithreitol (DTT), iodoacetamide, acetic acid, and formic acid were from Sigma (Zwijndrecht, The Netherlands). Titanium oxide was a gift from GL-Sciences (GLSciences, Inc., Japan). Zorbax extend (5 µm) (Agilent, Waldbronn, Germany) resin was used for the trap column, and ReproSil-Pur C18-AQ, 3 µm 120 Å (Dr. Maisch, HPLC GmbH, Ammerbuch-Entringen, Germany) resin was used for the analytical column. HPLC grade ACN was purchased from Biosolve (Valkenswaard, The Netherlands). Sample Preparations. For the test protein mixtures, serum albumin, alpha and beta casein, and hemoglobin (100 µM) were reduced in 1 mM DTT and alkylated in 2 mM iodoacetamide, following digestion with trypsin overnight at a protein/protease ratio of 50:1. On the day of analysis, the test mixture was prepared by dilution of the protein digests (100 µM) to 20 fmol/ µL in 0.5% formic acid. Human embryonic kidney (HEK) 293T cells were grown to confluence in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Invitrogen) and 0.05 mg/mL penicillin/ streptomycin (Invitrogen). Cells were washed with cold phosphate-buffered saline (PBS). Cells were consequently centrifuged at 1500 rpm to allow removal of the supernatant. Cells were lysed in 100 µL of 8 M urea and 50 mM ammonium bicarbonate, containing 5 mM sodium phosphate, 1 mM potassium fluoride, and 1 mM sodium orthovanadate, pH 8.2. Cellular debris was pelleted by centrifugation at 14 000 rpm for 20 min. Approximately 150 µg of protein material was used for analysis. Proteins were reduced with 1 mM DTT and alkylated with 2 mM iodoacetamide. The mixture was diluted 4-fold to 2 M urea using 100 µL of 50 mM ammonium bicarbonate and 50 µL of trypsin solution, 0.1 mg/mL, and incubated overnight at 37 °C. Strong Cation Exchange. Strong cation exchange (SCX) was performed using a Zorbax BioSCX-Series II column (0.8 mm (i.d.) × 50 mm (l), 3.5 µm), a FAMOS autosampler (LC-packing, Amsterdam, The Netherlands), a Shimadzu LC-9A binary pump, and a SPD-6A UV-detector (Shimadzu, Tokyo, Japan). Prior to SCX chromatography, protein digests were desalted using a small plug of C18 material (3 M Empore C18 extraction disk) packed into a GELoader tip (Eppendorf) similar to what has been previously described,40 onto which ∼10 µL of Aqua C18 (Phenomenex, Torrance, CA) (5 µm, 200 Å) material was placed. The eluate was dried completely and subsequently reconstituted in 20% ACN and 0.05% formic acid. After injection, a linear gradient of 1% min-1 solvent B (500 mM KCl in 20% ACN and 0.05% formic acid, pH 3.0) was performed. A total of 24 SCX fractions (1 min each, i.e., 50 µL elution volume) were manually collected and dried in a vacuum centrifuge. TiO2 Chips. UV laser ablation in combination with vacuum lamination of polyimide films was used to create the multilayer polymer based µ-fluidic devices. The starting material used was a polyimide film with good chemical resistance to solvents and 1566

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Mohammed et al. biofriendly to proteins and peptides. In a first step, the laser beam is focused on the polyimide film surface, and by variation of the energy per pulse, the laser spot size, and the cutting speed, one can vary width and depth of the channels created. The laser ablated films are aligned very precisely to each other and are then vacuum-laminated under temperature and pressure to form the monolithic-like fused chip. UV laser ablation is also used for 3D trimming of the laminated chip to form an integral electro-spray tip. Patterned noble metals are applied by thin film deposition on the polymer film surfaces to create electrical contacts for electrospray biasing. Finally, sections of the open microchannels are used for packing, which in our case are either trapping or enrichment sections or the analytical sections. For clarity in describing chip component descriptions and in order to allow a nomenclature similar to that of regular nanoLC, the terms precolumn and analytical column will be utilized for filled channels, and in the case of the precolumn, the term section will be applied to its subdivided compartments. The MS-mounted chip handler (commonly referred to as the ‘chip-cube’) allows mounting and positioning of the chip tip. The chip-cube also provides ultra low dead volume valving for flow path-switching. The microfluidic device is identified by an integrated RF-Tag, and all hydraulic connections are made by the system according to chip and application type. Two precolumn designs were investigated. 1. Design 1: ‘Two Sectioned’ Precolumn. On the basis of the single precolumn-analytical column design which was first described in ref 14, a common precolumn bed for reverse phase and TiO2 was created for the chip with a total volume of 320 nL(Figure 1). To achieve equal and accurate packing material volumes, a second chip that contained a 160 nL section was filled and used as the material reservoir for packing the ‘two sectioned’ precolumn. In a first step, RP material (Zorbax Extend 5µm, 160 nL) was filled into the common channel and then the rest of the channel was filled with TiO2 10 µm spheres (160 nL). The analytical channel (15 cm, 75 µm) was filled with Reprosil C18 material. 2. Design 2: Three Discrete Sectioned Precolumn. From our earlier work, we concluded that a three discrete sectioned precolumn as described31,41 could be advantageous to implement on a microfluidic device as well. To create three discrete sections for the precolumn, 2 additional layers were required. The two RP trapping sections of the precolumn were in a distinct location on one layer, while the TiO2 was on a separate layer with an intermediate layer providing separation. Liquid transfer was achieved through near-zero dead volume µ-sieve frit filters formed by UV laser ablation in the intermediate layers (Figure 2). Approximately 100 single micrometer holes are forming one thin film sieve frit µ-filter. The two end channels were individually filled with Zorbax Extend C18 (100 nL), while the center section was filled with TiO2 (45 nL). The analytical channel (15 cm, 75 µm) was filled with Reprosil C18 material. Online Enrichment and Analysis. Trapping, enrichment, and analysis was performed on an Agilent 1100 HPLC system, consisting of a loading pump operating in the microliter per minute (µL/min) range and an analytical pump operating in the nanoliter per minute (nL/min) range. Two Sectioned Precolumn. Peptides were trapped at 3 µL/ min using 100% solvent A (0.6% acetic acid). (Note: phosphorylated peptides will bind to the TiO2 section (enrichment), while all other peptides would be trapped on the subsequent C18 section.) An initial analysis was performed of what is trapped on the C18 section by switching the precolumn to be

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Figure 1. (a) Schematic of the TiO2-RP HPLC-chip having a common precolumn bed for TiO2 and RP. Total volume of the precolumn section is 320 nL (160 nL + 160 nL). The analytical column is 15 cm in length with an approximately rectangular cross section with a diagonal of 75 µm. (b) 3D zoom of the actual design which is to scale of the dual phase precolumn. (c) TiO2-RP HPLC-chip analysis of BSA, alpha casein, and hemoglobin (50 fmol each). The flow-through analysis where the uppermost panel is the BPI chromatogram, while the lower panels represent extracted ion chromatograms for the main phosphopeptides. (d) Chromatograms of what bound and eluted from the TiO2 enrichment column. All panels are extracted ion chromatograms for the indicated sequences.

in-line with the analytical column and nanoflow (analytical) pump (gradient from 5 to 40% solvent B (80% acetonitrile (ACN), 0.6%acetic acid) over 40 min at 200 nL/min; total analysis time 60 min). Elution of phosphorylated peptides from the TiO2 section to the C18 section was achieved by injection of 30 µL of 250 mM ammonium bicarbonate solution, pH 9.0 (adjusted with ammonia), containing 10 mM sodium phosphate, 5 mM sodium orthovanadate, and 1 mM potassium fluoride. Immediately afterward, an injection of 20 µL of 10% formic acid was performed to wash and re-equilibrate the precolumn. Analysis of the eluted (phosphorylated) peptides was performed by switching the precolumn to be in-line with the analytical column for a second H2O/ACN gradient. Three Sectioned Precolumn. Peptides were trapped at 3 µL/ min using 100% solvent A (0.6% acetic acid and 0.5% formic acid in water) on the first 100 nL C18 section. An initial analysis was performed by switching the precolumn to be in-line with the analytical column and nanoflow pump, followed by a gradient from 5 to 40% solvent B (80% ACN, 0.6% acetic acid, and 0.5% formic acid) over 40 min at 200 nL/min; total analysis time 60 min. (Note: phosphorylated peptides will bind to the TiO2 section when introduced; the low flow rate allows better conditions for binding when compared with direct loading of the sample onto a TiO2 section at a flow rate of 3 µL/min.) All other peptides, with no TiO2 affinity were chromatographically separated at ∼200 nL/min. Elution of phosphorylated peptides was achieved by injection of 30 µL of 250 mM ammonium bicarbonate solution, pH 9.0 (adjusted with ammonia), con-

taining 10 mM sodium phosphate, 5 mM sodium orthovanadate, and 1 mM potassium fluoride. Immediately afterward, an injection of 20 µL of 10% formic acid was performed to wash and re-equilibrate the precolumn. Analysis of the eluted (phosphorylated) peptides was performed by switching the precolumn to be in-line with the analytical column for a second H2O/ACN gradient. Mass Spectrometry. An Agilent MSD XCT+ ion trap mass spectrometer (Agilent, Walbronn, Germany) was operated in data-dependent mode, automatically switching between MS and MS/MS. MS spectra (from m/z 450–1500) were the average of 2 scans using a target value of 500 000. The three most intense ions (3 scan averages) were selected for collisioninduced fragmentation using a target value of 500 000 and a fragmentation amplitude of 1 V (with smart frag. 30%-200%). Data Analysis. Mascot generic files were generated from raw data using default values in Distiller (version 2.1.1.0 Matrix Science, U.K.). These peak lists were searched using an inhouse-licensed Mascot search engine (version 2.1.0, Matrix Science, U.K.). Files originating from the standard protein mixture analyses were searched against Swiss-Prot database (version 53.2), while those related to the HEK293 cells were against IPI Human (version 3.28). Carbamidomethyl cysteine was set as a fixed modification, while protein N-acetylation, oxidized methionines, and phosphorylation of serine and threonine were set as variable modifications. Trypsin was specified as the proteolytic enzyme and 1 missed cleavage was allowed. The mass tolerance of the precursor ion was set to Journal of Proteome Research • Vol. 7, No. 4, 2008 1567

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Figure 2. (a) Schematic of the RP-TiO2-RP ‘sandwich’ HPLC-chip: The graphic highlights the three discrete sections for precolumn RP (100 nL)-TiO2 (45 nL)-RP (100 nL) and 15 cm, 75 µm analytical column. (b) 3D zoom of the actual design of the precolumn indicating flow path during loading of sample; note design only allows analysis in forward flush mode. (c) Cross section of precolumn indicating the use of 6 layers in order to achieve the discrete precolumn sections. Green squares indicate zero dead volume links. (d) RP-TiO2-RP ‘sandwich’ HPLC-chip analysis of BSA, alpha casein, and hemoglobin (50 fmol each). The flow-through analysis where the uppermost panel is the BPI chromatogram, while the lower panels represent extracted ion chromatograms for the main phosphopeptides. (e) Chromatograms of what bound and eluted from the TiO2 enrichment column. All panels are extracted ion chromatograms for the indicated sequences.

400 ppm, and that of fragment ions was set to 0.9 Da. The threshold for MASCOT identification was set to 41 which corresponded to p < 0.01. Data is available as a zipped scaffold file at: https://bioinformatics.chem.uu.nl/supplementary/mohammed_JPR/.

Results Our starting point for chip design was the online TiO2 phosphopeptide enrichment system developed by Pinkse et al.14 The essential part of that design was a two sectioned precolumn where TiO2 preceded a regular C18 trapping section. During sample loading, phosphorylated peptides would bind to the TiO2 section, while all other peptides would flow through and be trapped on the C18 section. Figure 1 provides an overview of the two sectioned TiO2-RP precolumn chip. To keep the design as close to the robust 1D chip design,37 the channel that would become the precolumn was simply enlarged to allow 320 nL of packing material. In a first step, a well-defined volume of RP material (160 nL) was packed and then the rest of the chip channel was filled with TiO2 10 µm spheres. Initial testing of the design was performed with 50 fmol of bovine serum albumin, alpha casein, and hemoglobin digest. Loading of sample was performed at a ‘typical’ flow rate of 3 µL/min where phosphopeptides selectively bind to the first section of the precolumn, the TiO2, while the remaining peptides just flow through and concentrate on the C18 section 1568

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of the precolumn. An analysis of what was bound to the C18 was performed, which allowed an evaluation of the enrichment efficiency of the phosphopeptides to the TiO2. Subsequently, elution was performed with a pH 9 ammonium bicarbonate solution containing sodium phosphate, sodium orthovanadate, and 1 mM potassium fluoride. These Lewis base additives have been found to improve the efficiency of elution in titanium enrichment experiments and thus reduce the need to use more harsh conditions such as higher pH.31,41 A final injection of 10% FA was then performed to remove any residual elution buffer followed by a gradient analysis of the eluted peptides. Figure 1 represents extracted chromatograms for the main phosphorylated alpha casein peptides alongside the base peak chromatogram for both the flow-through and elution analysis. As can be clearly observed, efficient binding and elution of the phosphopeptides was achieved. Although, as the BPI chromatograms exhibits, a number of nonphosphorylated peptides are also present in the elution step. Further analysis of these peptides indicated they were highly acidic in nature. Contamination with acidic peptides represents a common issue in phosphopeptide enrichment based on metal co-ordination.11 However, in the case of TiO2, it was shown that acidic peptides show different binding behavior to phosphorylated peptides, and thus, this difference in conduct can be exploited to improve selectivity.15 A more pressing concern to arise with this two sectioned precolumn design was the reduction in enrichment efficiency over time of the TiO2 chip. On average, the percent-

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Figure 3. An on-chip enrichment/analysis of SCX fraction 7 from a 150 µg HEK293 cell lysate digest. (Left panels) BPI chromatogram represents the flow-through analysis (top), BPI chromatogram represents the elution analysis (bottom). (Right panels) Mass spectrum corresponding to the survey scan used to isolate and fragment GNIETTSEDGQVPpSPK (top), tandem mass spectrum of indicated peptide (bottom).

age of phosphopeptide that did not bind to the TiO2 was nearly 50% after only 10 analyses. Possible rationales for such poor retention are compromise of the TiO2 section by components present in the injected analytes as well as material that may originate from the autosampler itself such as metal and silica. Another major issue was that the efficiency of elution became poorer with each analysis, creating the necessity to perform multiple elutions. Our main suspicion for this multiple elution problem was that phase mixing could be occurring in the precolumn, as there were no discrete independent sections for the TiO2 and RP materials, and furthermore, the TiO2 particles are not uniform in size (Supplemental Figure 1 in Supporting Information). Although the flow through the precolumn is in the same direction during both the loading and analysis of sample, this section will experience quick flow speed and pressure changes when switching between the two modes. Such rapid changes can cause agitation and dislodging of the smaller TiO2 particles allowing them to migrate into the RP section. Such phase mixing can easily explain the precolumn degradation in a reused chip. To solve the issues that arose from this two sectioned precolumn, a new chip was developed, along the lines of the successful ‘sandwich’ chip design for online TiO2 enrichment we described previously.31,41 The ‘sandwich’ refers to an additional RP section placed in front of the TiO2 section which will behave as a guard and allow binding of phosphopeptides to the TiO2 particles to be more efficient, that is, an RP-TiO2RP precolumn. The chip was increased to a 6 layer design to accommodate discrete regions for each section of the ‘precolumn’, thereby eliminating any issues caused by phase mixing (Figure 2a-c). The size of the TiO2 section was also reduced to improve ability to elute. This is possible because the TiO2 has a far higher capacity than the RP material.15 An additional consequence of this design is that all peptides, including those that are phosphorylated, will bind initially to the first RP section. An analysis of what is bound to the first section of the precolumn will allow the phosphopeptides to migrate and bind to the TiO2 section, while all other peptides will continue onto the analytical column for separation and MS detection. Figure 2d,e demonstrates typical performance of the new design. What is noteworthy is the chromatograms displayed in Figure 2 represent analysis number 30 on that particular chip. Thus, the 3-discrete sections of the precolumn eliminate issues relating

to poor binding and elution. The issue relating to selectivity of phosphopeptides and coenrichment of acidic peptides raised concerns about efficacy of the online enrichment approach. However, dramatic improvements in selectivity were achieved through the use of a modest amount of formic acid (0.5%) in the LC solvents as exhibited in Figure 2e where the phosphopeptides have become the base peaks in the elution analysis.31,42 Although the formic acid improves selectivity, one can imagine the requirement to attain an additional level of improvement that may only be possible through the use of additives such as dihydroxybenzoic acid15 or lactic acid.43 It is also possible to implement the use of additives into the online chip TiO2 system by performing ‘wash’ steps, while the phosphopeptides are retained on the TiO2 section before the elution step. Ideal applications for online enrichment analysis are cellular phosphoproteome studies. A typical method for performing such an experiment is the use of SCX fractionation at low pH to perform an initial enrichment of phosphorylated peptides18 followed by a second step of enrichment based on IMAC27 or TiO2.16,44 The SCX step will produce a large number of fractions (usually numbering around 20–50), and so, an automated online secondary enrichment step would be highly desirable. To test the applicability of the sandwich TiO2 chip, an SCXTiO2 experiment using 150 µg of human embryonic kidney (HEK) 293T cell lysate was performed. A new chip was used, and after an initial analysis of the standard phosphopeptide, it was ready for use. Figure 3a,b represents the base peak intensity chromatograms for the flow-through and elution analysis of the online TiO2 enrichment for one of these (HEK) 293T SCX fractions. As the figure highlights, the amount of material present in the flowthrough is dramatically higher than the elution indicating a significant level of reduction in sample complexity. Furthermore, a detailed analysis of this TiO2 analysis shows mostly N-acetylated peptides and protein C-termini in the flow-through as well as a few phosphorylated peptides (all of which are present in the elution), while over 80% of peptides present in the elution fraction were phosphorylated. Over 100 phosphopeptides were found in this single 60 min gradient (90 min, total) analysis of this fraction. All data can be viewed through a download of a scaffold file found at https://bioinformatics.chem.uu.nl/supplementary/mohammed_JPR/. However, the representation of phosphorylated peptides in the elution can be improved through the use of MS3 triggered Journal of Proteome Research • Vol. 7, No. 4, 2008 1569

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neutral loss scans, multistage activation, or an instrument with better mass accuracy, resolution, and more comprehensive CAD such as a quadrupole-time-of-flight (Q-ToF) instrument which will improve sequencing of these awkward species and create a more realistic picture of enrichment levels. During the analysis of these SCX fractions, three additional analyses of the standard proteome mixture were performed (Supplementary Figure 2 in Supporting Information). Beta casein was added to the standard mixture to provide the phosphopeptide FQpSEEQQQTEDELQDK which is known to have a high affinity (and therefore strong retention) for TiO2 and other metal based methods due to its highly acidic nature alongside its phosphate moiety. Such a peptide will highlight issues relating to efficient elution from TiO2. Supplemental Figure 2 (Supporting Information) contains the three additional elution analyses performed during the use of the new design titanium chip and highlights a consistent level of performance over 20 analyses. To gauge reproducibility of the sandwich TiO2 chip, the initial analysis of the standard was used as a reference for the subsequent robustness checks. The intensities observed in this initial elution, that is, first analysis, were deemed to represent ‘100%’ efficiency. When the extracted ion chromatograms across these 3 additional ‘standard’ analyses were used, the phosphopeptide signal average was 150.6% ((62.4%). The standard deviation across only the 3 additional analyses was 22% which follows expectation since the chip requires at least one full analysis cycle to become consistent. Additionally, the retention time of these peptides differed by less than 10 s on a 60 min analysis. Furthermore, the only peptide that was observed (once) in the flow-through analysis was YKVPQLEIVPNpSAEER. This was not unsurprising since mis-cleavage peptides are known to have poorer binding efficiencies.45 This initial sandwich RP-TiO2RP chip proved more than able for an SCX-TiO2 experiment with the chip providing a consistent level of performance across the analyses with little or no intervention from the operator.

Concluding Remarks An automated online TiO2 chip based liquid chromatographic approach, aimed at enriching phosphorylated peptides from large complex proteolytic digests, is described. By employing an RP-TiO2-RP ‘sandwich’ precolumn, as successfully described for an online capillary approach,31,41 a facile and sensitive device was developed that requires little intervention from the user to acquire successful operation. This three sectioned sandwich TiO2 precolumn chip allows the same high selectivity of phosphopeptide-enrichment as the capillary design but without the need for column preparation and expert handling. The chip can be simply inserted into the chip cube and is ready for analysis, overcoming typical experimental hurdles as dead-volumes and tubing artifacts. In these first chips, performance could be maintained over tens of analyses or days of operation with little change in phosphopeptide enrichment efficiency and analysis. Once the design can survive close to a hundred analyses, it can be a highly feasible technology for nonexperts to perform phosphoproteomics analysis. Improvements can still be envisaged, with maybe even longer analytical columns and the coupling of the microfluidic device with a more advanced mass spectrometer.31,41

Acknowledgment. This work was financially supported by The Netherlands Proteomics Centre (www.netherlandsproteomicscentre.nl) and the Agilent 1570

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Technologies foundation. We would also like to thank Georges Gauthier and Agilent Technologies for their assistance.

Supporting Information Available: Figures of the schematic representing the cross section TiO2-RP HPLC-chip precolumn; BPI and extracted chromatograms for three TiO2 elution analyses of a standard protein mixture; table of peak intensities and peptide retention times for four standard protein mixture analyses. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Jensen, O. N. Interpreting the protein language using proteomics. Nat. Rev. Mol. Cell Biol. 2006, 7 (6), 391–403. (2) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. The protein kinase complement of the human genome. Science 2002, 298 (5600), 1912. (3) Mann, M.; Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21 (3), 255–261. (4) Linding, R.; Jensen, L. J.; Ostheimer, G. J.; van Vugt, M.; Jorgensen, C.; Miron, I. M.; Diella, F.; Colwill, K.; Taylor, L.; Elder, K.; Metalnikov, P.; Nguyen, V.; Pasculescu, A.; Jin, J.; Park, J. G.; Samson, L. D.; Woodgett, J. R.; Russell, R. B.; Bork, P.; Yaffe, M. B.; Pawson, T. Systematic discovery of in vivo phosphorylation networks. Cell 2007, 129 (7), 1415–1426. (5) Steen, H.; Jebanathirajah, J. A.; Rush, J.; Morrice, N.; Kirschner, M. W. Phosphorylation analysis by mass spectrometry-Myths, facts, and the consequences for qualitative and quantitative measurements. Mol. Cell. Proteomics 2006, 5 (1), 172–181. (6) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann, M.; Lodish, H. F. Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sc. U.S.A. 2000, 97 (1), 179–184. (7) Gronborg, M.; Kristiansen, T. Z.; Stensballe, A.; Andersen, J. S.; Ohara, O.; Mann, M.; Jensen, O. N.; Pandey, A. A mass spectrometrybased proteomic approach for identification of serine/threoninephosphorylated proteins by enrichment with phospho-specific antibodies - Identification of a novel protein, Frigg, as a protein kinase A substrate. Mol. Cell. Proteomics 2002, 1 (7), 517–527. (8) Zheng, H. Y.; Hu, P.; Quinn, D. F.; Wang, Y. K. Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography. Mol. Cell. Proteomics 2005, 4 (6), 721– 730. (9) Posewitz, M. C.; Tempst, P. Immobilized gallium(III) affinity chromatography of phoshopetides. Anal. Chem. 1999, 71 (14), 2883–2892. (10) Stensballe, A.; Andersen, S.; Jensen, O. N. Characterization of phosphoproteinhs from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 2001, 1 (2), 207–222. (11) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20 (3), 301–305. (12) Kweon, H. K.; Hakansson, K. Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. Anal. Chem. 2006, 78 (6), 1743–1749. (13) Chen, C. T.; Chen, W. Y.; Tsai, P. J.; Chien, K. Y.; Yu, J. S.; Chen, Y. C. Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis. J. Proteome Res. 2007, 6 (1), 316–325. (14) Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 2004, 76 (14), 3935–3943. (15) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 2005, 4 (7), 873–886. (16) Benschop, J. J.; Mohammed, S.; O’Flaherty, M. C.; Heck, A. J. R.; Slijper, M.; Menke, F. L. H. Quantitative phospho-proteomics of early elicitor signalling in Arabidopsis. Mol. Cell. Proteomics 2007, 6 (7), 1198–1214.

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PR700635A

Journal of Proteome Research • Vol. 7, No. 4, 2008 1571