Enrichment of Phosphopeptides Using Biphasic Immobilized Metal

Jul 22, 2008 - Abstract: Immobilized metal affinity chromatography. (IMAC) based on Fe3+ or Ga3+ chelation is the most widely employed technique for t...
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Enrichment of Phosphopeptides Using Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns Michael Schilling and Daniel R. Knapp* Department of Pharmacology and MUSC Proteomics Center, Medical University of South Carolina, Charleston, South Carolina, 29425 Received February 14, 2008

Abstract: Immobilized metal affinity chromatography (IMAC) based on Fe3+ or Ga3+ chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of β-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of β-casein monophosphorylated peptide in 20 µL of a solution of human serum protein digest (from 200 µg total serum protein after digestion and desalting). Keywords: IMAC • titanium dioxide • phosphopeptide enrichment

Introduction Protein phosphorylation is one of the most prominent and intensively studied post translational modifications in biological systems. Despite estimates that only 10-30% of cellular proteins are thought to be phosphorylated at any time,1,2 reversible protein phosphorylation remains a critical regulator of cellular events including proliferation, division, differentiation, signal transduction, and gene expression.3–7 Given the importance of phosphorylation, large-scale phosphoprotein analysis utilizing both MALDI and electrospray based mass spectrometry has emerged as the preferred technology due to its inherent high sensitivity, selectivity, and speed over conventional biochemical methods.2,8,9 Approaches for the determination of phosphorylation sites have primarily incorporated analysis of phosphopeptides via tandem mass spectrometry. However, large-scale characterization of phosphorylation fre* To whom correspondence should be addressed. E-mail: knappdr@ musc.edu.

4164 Journal of Proteome Research 2008, 7, 4164–4172 Published on Web 07/22/2008

quently is quite challenging due to both biological and analytical limitations. Substoichiometric phosphorylation of many proteins in combination with low abundance can lead to difficulties in identification. In addition, the presence of nonphosphorylated peptides can severely diminish phosphopeptide characterization presumably through ionization suppression in positive mode detection. Moreover, phosphopeptides tend to have lower ionization efficiencies due to an increase in negatively charged sites, especially for multiphosphorylated forms, and because of their hydrophilicity phosphopeptides may be subject to selective loss during routine sample preparation. Also, tandem mass spectrometric analysis of phosphopeptides can result in poor quality spectra as often the dominant loss of phosphoric acid impairs cleavage along the peptide backbone and, with it, vital sequence information.10–13 However, electron capture dissociation (ECD) and electron transfer dissociation (ETD) are emerging technologies which can overcome facile loss of phosphoric acid since fragmentation occurs predominately along the peptide backbone with phosphorylation site integrity remaining intact.14,15 To overcome sample complexity, phosphopeptide enrichment strategies have been developed to facilitate the investigation of the phosphoproteome. Strong-cation exchange chromatography (SCX) at low pH has been exploited to isolate tryptic phosphopeptides originating from the nuclear fraction of a HeLa cell lysate in early eluting fractions.16 Conversely, phosphopeptides from mouse liver were found to elute across the entire SCX gradient.17 Anion-exchange chromatography solely18 and in a mixed-bed format with SCX19 was utilized to selectively enrich phosphopeptides. Electrophoretic techniques have also been shown to be useful for the enrichment of phosphopeptides.20,21 For instance, Chong-Feng Xu et al.22 incorporated methyl esterification of protein digests to spread isoelectric points of phosphorylated and nonphosphorylated peptides in order to capture the phosphorylated species on focusing gels. Immunoaffinity approaches, particularly antibodies which recognize low abundance phosphotyrosine residues, have been employed in phosphopeptide enrichment.17,23 Chemical derivatization strategies, most commonly β-elimination alone24 or coupled to Michael addition for the capture of phosphopeptides with streptavidin via the incorporation of an affinity tag,25,26 have been reported. Thompson et al.27 integrated on-resin chemical derivatization of metal chelated phosphopeptides to facilitate their characterization by MALDIPSD/LIFT tandem mass spectrometry. Phosphoramidate chemistry has been utilized in an alternate covalent capture method 10.1021/pr800120f CCC: $40.75

 2008 American Chemical Society

technical notes

Enrichment of Phosphopeptides

Figure 1. MALDI-MS spectra of 20 pmol trypsin digested β-casein (a) without phosphopeptide enrichment, (b) enriched using IMAC (Pro-Q Diamond)-C18 biphasic microcolumn, and (c) enriched using TiO2 tip. Peaks marked with asterisks denote phosphopeptides. Enrichment significantly reduces spectral complexity through enhancement of phosphopeptide signals concurrent with suppression of nonphosphorylated peptide peaks. Table 1. Identified Phosphorylated Peptides from Tryptic Digestion of β-Casein and Ovalbumin Enriched via IMAC C18 Biphasic Microcolumns or TiO2 Tips peptide mass [M + H]+ (Da) peptide sequence (β-casein)a

degree of phosphorylation

IMAC-C18

TiO2

FQSEEQQQTEDELQDK (33-48) NVPGEIVESLSSSEESITR (22-40) ELEELNVPGEIVESLSSSEESITR (17-40) RELEELNVPGEIVESLSSSEESITR (16-40)

1 4 4 4

2064.80 2355.11 2969.53 3125.75

2063.89 2355.03 2968.98 3124.74

peptide mass [M + H]+ (Da)

a

peptide sequence (ovalbumin)a

degree of phosphorylation

IMAC-C18

TiO2

EVVGSAEAGVDAASVSEEFR (340-359) FDKLPGFGDSIEAQCGTSVNVHSSLR (59-84)

1 1

2091.73 2905.10

2090.97 2903.57

Bold amino acid residues denote phosphorylation sites.

employing matrices with immobilized iodoacetyl28 and maleimide groups29 as well as through the use of polyamine based dendrimers.30 However, these synthetic approaches have several handicaps including unwanted side reactions and potentially significant sample loss; therefore, they have not been used extensively in the characterization of protein phosphorylation. The most widely used phosphopeptide enrichment strategy to date has been immobilized metal affinity chromatography (IMAC). Despite its widespread popularity, IMAC suffers from the coenrichment of mainly acidic nonphosphorylated peptides containing glutamic and aspartic acid residues as well as peptides whose sequences contain histidine. The existence of nonphosphorylated peptides renders phosphopeptide detection and subsequent site identification more difficult due to ionization suppression effects. To reduce nonspecific binding to IMAC resins methyl esterification of C-terminal and internal

carboxyl groups has been introduced.31–33 However, incomplete methyl esterification of peptides containing multiple carboxyl groups results in complicated mass spectra. In addition, unwanted side reactions, including deamidation and subsequent methylation of asparginine and glutamine residues, further increase spectral complexity. Also, the variability in phosphopeptide selectivity/specificity and ultimate recovery via IMAC enrichment has been well documented.34–36 For example, Ndassa et al.34 showed the effect of subtle differences in experimental conditions (i.e., loading, washing, elution, and column capacity) on IMAC enrichment of phosphopeptides from K562 cell lysates. In a comparison of four commercially available IMAC products, discrepancies in the enrichment of tryptic digests of phosphoprotein mixtures alone and incorporated into a bovine serum albumin digest were reported.36 Haydon et al.37 illustrated that methyl esterification of a Journal of Proteome Research • Vol. 7, No. 9, 2008 4165

technical notes

Schilling and Knapp

Figure 2. MALDI-MS spectra of 20 pmol trypsin digested ovalbumin (a) without phosphopeptide enrichment, (b) enriched using IMAC (Pro-Q Diamond)-C18 biphasic microcolumn, and (c) enriched using TiO2 tips. Peaks marked with asterisks denote phosphopeptides.

Figure 3. MALDI-MS spectra of mixtures containing phosphorylated (β-casein and ovalbumin) and nonphosphorylated (bovine serum albumin and β-lactoglobulin) protein tryptic digests in varying ratios of (a) 1:1:1:1, (b) 1:1:10:10, and (c) 1:1:50:50 enriched via IMAC (Pro-Q Diamond) -C18 biphasic microcolumns. Peaks marked with asterisks denote phosphopeptides.

Xenopus laevis Aurora A protein digest resulted in a reduction of total protein coverage with concurrent nonspecific binding. However, selective recovery of phosphopeptides did increase two to 3-fold after methyl esterification. 4166

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Systematic investigations of both stationary phase chemistry and chelating metal employed in IMAC enrichment have been reported with inconsistent results.18,38–42 For instance, the capture and release of 32P-labeled Arabidopsis membrane

technical notes

Enrichment of Phosphopeptides Table 2. Identified Phosphorylated Peptides from Tryptic Digestion Mixtures Enriched via IMAC C18 Biphasic Microcolumns or TiO2 Tips peptide mass [M + H] (Da) peptide sequence

a

IMAC-C18

TiO2

1:1:1:1 FQSEEQQQTEDELQDK (β-cas) EVVGSAEAGVDAASVSEEFR (Ova) NVPGEIVESLSSSEESITR (β-cas) EVVGSAEAGVDAASVSEEFR (Ova) ELEELNVPGEIVESLSSSEESITR (β-cas) RELEELNVPGEIVESLSSSEESITR (β-cas)

2063.25 2089.71 2354.90 2903.80 2967.57 3124.53

2063.08 2355.98 2968.40 3125.46

1:1:10:10 FQSEEQQQTEDELQDK (β-cas) EVVGSAEAGVDAASVSEEFR (Ova) NVPGEIVESLSSSEESITR (β-cas) ELEELNVPGEIVESLSSSEESITR (β-cas) RELEELNVPGEIVESLSSSEESITR (β-cas)

2063.75 2090.91 2354.86 2968.46 3124.69

2063.04 2355.2 2968.45 3124.81

1:1:50:50 FQSEEQQQTEDELQDK (β-cas) EVVGSAEAGVDAASVSEEFR (Ova) ELEELNVPGEIVESLSSSEESITR (β-cas) RELEELNVPGEIVESLSSSEESITR (β-cas)

2062.85 2090.80 2968.46 3123.93

-

a

Bold amino acid residues denote phosphorylation sites.

phosphopeptides via IMAC incorporating a Fe3+-iminodiacetic acid (IDA) resin were found to be superior to ZrO2+-IDA and Fe3+-nitrilotriacetic acid (NTA) resins.18 Contradictory, Neville et al.38 showed Fe3+-NTA to retain phosphopeptides with higher specificity than Fe3+-IDA chelated IMAC resins. In addition, Posewitz et al.39 reported Ga3+-IDA being more selective in binding phosphopeptides than Fe3+-IDA. Selectivity toward monophosphorylated peptide isolation by iron chelated NTA and multiphosphorylated peptide capture by gallium chelated NTA has been suggested.40 More recently, the use of metal oxides, particularly titanium dioxide (TiO2), has shown great promise as an alternative to IMAC for the enrichment of phosphopeptides.43–46 However, similar to IMAC, binding of nonphosphorylated peptides has been found to occur with metal oxide enrichment. Additives which reduce nonspecific binding, but allow for phosphopeptide capture to titanium dioxide, have been incorporated including 2,5-dihydroxybenzoic acid (DHB),47 DHB combined with 1-octanesulfonic acid (OSA),48 phthalic acid,49 lactic acid,50 glycolic acid,51 and glutamate/glutamic acid.52,53 However, a decrease in sensitivity of phosphopeptide detection using LC-ESI-MS/MS with DHB and phthalic acid has been noted.48,50,53 Phosphopeptide selectivity/specificity and recovery via metal oxide enrichment have been shown to be very sensitive to experimental conditions.51,53 In a comparison of metal oxide based enrichment of phosphopeptides, Kweon et al.43 noted a bias toward singly phosphorylated peptide capture with zirconium dioxide (ZrO2) as opposed to preferential isolation of multiphosphorylated forms using titanium dioxide. Comparison of titanium dioxide and IMAC for phosphopeptide enrichment has been the subject of multiple reports.54–57 An online multidimensional LC-MS approach utilizing TiO2 for phosphopeptide enrichment was shown to be superior to IMAC and ZrO2 for tryptic digests of R- and β-caseins.54 However, this method was optimized for TiO2 and applied directly to IMAC/ZrO2, thereby, inherently introducing a bias toward successful TiO2 enrichment. Zhang et al.55 reported

improved phosphopeptide selectivity in a combined calcium phosphate precipitation-IMAC strategy versus subsequent capture by TiO2. Significant discrepancies in phosphopeptide selectivity have also been noted, namely preferential binding of monophosphorylated peptides to TiO2 and multiphosphorylated peptides to IMAC resins.18,37,51,55 Thingholm et al.56 introduced a sequential strategy for the separation of monophosphorylated and multiphosphorylated peptides based on pH from highly complex biological samples utilizing both titanium and IMAC enrichment. Secondarily, the influence of mass spectrometer settings, particularly neutral loss events, was shown to be highly biased toward detection of particular degrees of phosphorylation. The use of ECD or ETD fragmentation as discussed previously would circumvent the labile nature of phosphorylation when analyzed via collision induced dissociation. Also, a comparison of three phosphopeptide enrichment techniques including IMAC, TiO2, and phosphoramidate chemistry revealed capture of different, partially overlapping segments of the phosphoproteome from the cytosolic fraction of Drosophila melanogaster Kc 167 cells.57 Thus, it is clear that no single enrichment approach is efficiently global in scale for the complete coverage of the phosphoproteome. Therefore, in an attempt to further explore enrichment strategies for complex biological samples, we investigated a commercially available resin, Pro-Q Diamond, marketed mainly toward phosphoprotein enrichment, for isolation of phosphopeptides. Pro-Q Diamond resin is based upon a proprietary Pro-Q Diamond stain (Invitrogen) for detecting phosphoproteins on gels. The composition of the Pro-Q Diamond resin is proprietary, but based upon information provided in the patent58 and inductively coupled plasma (ICP) mass spectrometry analysis of the material, it has been identified as a chelated gallium IMAC resin. Here, we present a comparison of approaches using Pro-Q Diamond and TiO2 tips for the capture of phosphopeptides from tryptic digests of β-casein and ovalbumin as well as from synthetic mixtures of standard protein digests using biphasic IMAC-reversed phase microcolumns.59,60 This work to our knowledge is the first report of a direct comparison of Pro-Q Diamond resin, which according to patent literature58 appears to be based on (1,2-bis (2aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid chemistry (BAPTA), with titanium dioxide for the enrichment of phosphopeptides. Other reports utilizing both IMAC and titanium dioxide have incorporated resins comprising metal chelating moieties based on IDA54 or NTA55,56 chemistry. In addition, the sensitivity of the method is probed using β-casein monophosphopeptide spiked human serum.

Experimental Section Materials. Bovine β-casein, bovine β-casein monophosphorylated peptide (FQSEEQQQTEDELQDK), chicken ovalbumin, bovine serum albumin (BSA), β-lactoglobulin, iodoacetamide, proteomics grade trypsin, 2,5-dihydroxybenzoic acid (DHB), ammonium hydroxide, a bicinchoninic acid (BCA) kit, and human serum were obtained from Sigma-Aldrich (St Louis, MO). Ammonium bicarbonate, urea, acetic acid, phosphoric acid, HPLC-grade water, and HPLC-grade acetonitrile were obtained from Fisher Scientific (Fair Lawn, NJ). Formic acid and dithiothreitol were obtained from Fluka (Buchs, Switzerland). Sep-Pak tC18 solid-phase extraction cartridges (1 mL) were obtained from Waters (Milford, MA), µ-C18 Ziptips from Millipore (Billerca, MA), and 200 µL titanium dioxide tips from Glygen (Columbia, MD). Pro-Q Diamond resin was obtained Journal of Proteome Research • Vol. 7, No. 9, 2008 4167

technical notes

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Figure 4. MALDI-MS spectra of mixtures containing phosphorylated (β-casein and ovalbumin) and nonphosphorylated (bovine serum albumin and β-lactoglobulin) protein tryptic digests in varying ratios of (a) 1:1:1:1, (b) 1:1:10:10, and (c) 1:1:50:50 enriched via TiO2 tips. Peaks marked with asterisks denote phosphopeptides.

as a component of a protein enrichment kit from Invitrogen (Carlsbad, CA). Digestion of Phosphoprotein Standards, Synthetic Mixtures, and Human Serum. Fifty microliters of a 1 µg/µL stock solution of β-casein or ovalbumin were diluted with 450 µL of 20 mM ammonium bicarbonate. For β-casein, trypsin was added in a 1:20 (enzyme:substrate) ratio and incubated overnight at 37 °C with constant agitation. In the case of ovalbumin, reduction of disulfide bonds was carried out in 10 mM dithiothreitol at 60 °C for one hour followed by alkylation in 30 mM iodoacetamide at ambient temperature in darkness for 30 min. Finally, trypsin was added in a 1:20 (enzyme:substrate) ratio and incubated overnight at 37 °C with constant agitation. Digestion of bovine serum albumin and β-lactoglobulin was achieved as described above for β-casein. All digested samples were stored at -20 °C. One milligram of human serum protein (original human serum protein concentration determined to be 95 mg/ mL according to BCA protein assay) was denatured in 7.8 M urea followed by reduction in 10 mM dithiothrietol at 37 °C for 1 h in darkness. Subsequently, alkylation was performed in 30 mM iodoacetamide at 37 °C for one hour in darkness. The solution was then diluted to 1.85 M urea using 200 mM ammonium bicarbonate, 20 µg of trypsin added, and incubated overnight at 37 °C. Following digestion, the sample was desalted via C18 solid phase extraction, and peptides eluted with 1.0 mL of 0.1% formic acid/70% acetonitrile. Phosphopeptide Enrichment Via Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns and Titanium Dioxide Tips. Phosphoprotein Standard Digests. Biphasic immobilized metal affinity-reversed phase microcolumns were constructed through the addition of Pro-Q Diamond resin slurry to µ-C18 Ziptips resulting in IMAC bed heights of 6-7 mm. One hundred microliters of 0.01% acetic acid was passed through the column in 20 µL increments using gentle pressure 4168

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created by the application of a 5 mL plastic syringe to the top of the tip. All subsequent steps were performed in the same manner. The column was then equilibrated with 100 µL of 70 mM acetic acid/50% acetonitrile (loading buffer), and 20 pmol of β-casein digest dissolved in 20 µL of 70 mM acetic acid/ 50% acetonitrile were loaded onto the column using five repetitive cycles (i.e., single 20 µL aliquot of 20 pmol β-casein digest passed over column five times). Subsequently, residual nonphosphorylated peptides were removed by washing the column with 100 µL of loading buffer followed by re-equilibration of the C18 phase with 100 µL of 0.1% formic acid. IMAC captured phosphopeptides were then transferred to the reversed phase material by passing 20 µL of 1% phosphoric acid over the column using five repetitive cycles,59,60 and any remaining phosphoric acid was eliminated by washing the column with 100 µL of 0.1% formic acid. Finally, phosphopeptides were eluted from the C18 phase via 2 µL of 0.1% formic acid/50% acetonitrile directly onto a MALDI target plate. After partial evaporation of the drop, 2 µL of 20 mg/mL DHB in 80% acetonitrile/1% phosphoric acid was added. The addition of 2 µL of DHB matrix to the partially evaporated sample spot was repeated to ensure favorable crystallization. Inclusion of 1% phosphoric acid to DHB matrix has been shown to substantially improve phosphopeptide detection by MALDI.61 Ovalbumin phosphopeptides were isolated in the same fashion except 20 pmol digests were desalted using µ-C18 Ziptips prior to biphasic microcolumn enrichment. Each column was relegated to single use to avoid cross contamination. Titanium dioxide enrichment was performed according to Cantin et al.54 with slight modifications. Briefly, titanium dioxide tips attached to a 200 µL Pipetman (Gilson, Middleton, WI) were equilibrated with 2% formic acid/30% acetonitrile using ten 150 µL aspirate/dispense cycles (i.e., collection of solution in the tip followed by immediate ejection from the

Enrichment of Phosphopeptides

technical notes

Figure 5. LC-MS base peak chromatograms of trypsin digested human serum spiked with (a) 20 pmol, (b) 10 pmol, (c) 5 pmol, (d) 1 pmol, and (e) 0.2 pmol of β-casein monophosphopeptide standard and subsequently enriched using IMAC (Pro-Q Diamond) -C18 biphasic microcolumns.

tip). Twenty picomoles of β-casein or ovalbumin digest dissolved in 150 µL of 2% formic acid/30% acetonitrile were then loaded onto titanium dioxide tips via fifty 150 µL aspirate/ dispense cycles. The tips were subsequently washed with 2% formic acid/80% acetonitrile using ten 150 µL aspirate/dispense cycles. Finally, phosphopeptides were eluted with eight 25 µL aliquots of 0.5% ammonium hydroxide and dried in a vacuum centrifuge. Phosphopeptides were subsequently dissolved in 2 µL of 20 mg/mL DHB in 80% acetonitrile/1% phosphoric acid and spotted onto a MALDI target plate. Synthetic Mixtures. Phosphopeptides were enriched with biphasic columns and titanium dioxide tips from mixes of phosphorylated (β-casein and ovalbumin) and nonphosphorylated (bovine serum albumin and β-lactoglobulin) protein standard digests as described above in the following ratios (amounts): 1:1:1:1 (2 pmol β-casein: 2 pmol ovalbumin: 2 pmol BSA: 2 pmol β-lactoglobulin), 1:1:10:10 (2 pmol β-casein: 2 pmol ovalbumin: 20 pmol BSA: 20 pmol β-lactoglobulin), and 1:1: 50:50 (2 pmol β-casein: 2 pmol ovalbumin: 100 pmol BSA: 100 pmol β-lactoglobulin). Human Serum Digest. Following Sep-Pak desalting, the 1.0 mL volume of eluent containing digested human serum (from 1 mg total serum protein) was aliquoted into five 200 µL fractions and dried in a vacuum centrifuge. Each aliquot was then reconstituted in 20 µL of 70 mM acetic acid/50% acetonitrile, spiked with varying amounts of β-casein monophos-

phopeptide (0.2 - 20 pmol), and enriched via biphasic immobilized metal affinity-reversed phase microcolumns as described above. The phosphopeptide eluents were dried in a vacuum centrifuge and each subsequently solubilized in 3 µL of 0.1% formic acid for LC-MS3 analysis. Mass Spectrometry. MALDI-TOF MS. MALDI MS spectra were acquired using a Bruker Autoflex III time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) with positive ion linear mode detection. Each sample spot was interrogated with a 300 shot count. Reversed Phase LC-ESI-MS3. Biphasic column enriched β-casein monophosphopeptide spiked human serum samples were loaded directly onto a 15 cm × 75 µm RP-C18 capillary column (Micro-Tech Scientific, Vista, CA) via a 1 µL sample loop. Peptides were separated with a 40 min linear gradient of 100% mobile phase A (98% H2O/2% acetonitrile/0.1% formic acid) to 70% mobile phase B (2% H2O/98% acetonitrile/0.1% formic acid) at a flow rate of 180 nL/min using an Ultimate nanoflow LC system (Dionex, Sunnyvale, CA). Eluted peptides were detected in the positive ion mode by an LTQ mass spectrometer (Thermo Scientific, Waltham, MA) fitted with a nanospray source. The mass spectrometer was programmed in the data-dependent mode for neutral loss detection of phosphoric acid. Specifically, MS/MS analysis was performed on the five most intense ions resulting from the first stage MS spectrum scanned over m/z 400-2000. MS/MS/MS analysis Journal of Proteome Research • Vol. 7, No. 9, 2008 4169

technical notes was subsequently performed only if a neutral loss event of phosphoric acid (m/z 98, 80, 49, 40, 32.7) from collision induced dissociation (CID) was detected from the three most intense ions observed in the MS/MS spectrum. Mass lists included the doubly (m/z 1032) and triply (m/z 688) charged ions expected to result from the monophosphopeptide of β-casein with a window of (2 Da. Dynamic exclusion was enabled for all analyses with a repeat count of 2, a repeat duration of 30 s, an exclusion duration of 180 s, and a rejection mass width of 3 Da. A nanospray voltage of 2.4 kV and a normalized collision energy of 35% were used throughout.

Results and Discussion Comparison of Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns and Titanium Dioxide Tips for the Enrichment of Phosphopeptides from Protein Standards. The efficiency of an optimized biphasic immobilized metal affinityreversed phase microcolumn approach in comparison to titanium dioxide for the selective enrichment of phosphopeptides was evaluated using tryptic peptides originating from β-casein and ovalbumin. Trypsin digestion of β-casein is expected to produce three phosphorylated fragments: a monophosphorylated peptide with a m/z value of 2063 and two tetraphosphorylated peptides characterized by m/z values of 2967 and 3124, respectively, due to missed tryptic cleavages.62 The MALDI mass spectrum of 20 pmol unenriched digested β-casein is shown in Figure 1a. Despite the lack of phosphopeptide enrichment, the primary expected phosphopeptides for β-casein were nevertheless detected using the DHB matrix doped with 1% phosphoric acid. The m/z values for detected phosphopeptides (denoted by an asterisk) are listed in Table 1. One additional reported variation of the tetraphosphorylated peptide was detected with a m/z value of 2355 in the unenriched digest.51 The MALDI mass spectrum for the biphasic immobilized metal affinity-reversed phase microcolumn enrichment of 20 pmol digested β-casein is shown in Figure 1b. Both the monophosphorylated and tetraphosphorylated peptides are detected with an increased signal intensity for the tetraphosphorylated forms at higher m/z values. Also, signals resulting from nonphosphorylated peptides have been significantly reduced upon enrichment. Titanium dioxide enrichment (Figure 1c) of 20 pmol digested β-casein also shows an increase in the signal intensity of the tetraphosphorylated peptides. However, compared to the biphasic microcolumn approach titanium dioxide capture appears to coenrich a slightly higher number of nonphosphorylated peptides. Trypsin digestion of ovalbumin is expected to result in three monophosphorylated peptides with m/z values of 2091, 2513, and 2903 respectively.63 Figure 2a shows the MALDI mass spectrum of 20 pmol unenriched digested ovalbumin. Two of the monophosphorylated peptides are detected using the DHB matrix doped with 1% phosphoric acid. The m/z values for the detected phosphopeptides (denoted by an asterisk) are listed in Table 1. Upon enrichment of 20 pmol digested ovalbumin via biphasic immobilized metal affinity-reversed phase microcolumns, a substantial increase in the signal intensity of both monophosphorylated peptides with a concurrent suppression of peaks corresponding to nonphosphorylated peptides can be seen in Figure 2b. Similar results were obtained through titanium dioxide enrichment as illustrated in the MALDI mass spectrum of Figure 2c. However, the monophosphorylated 4170

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Schilling and Knapp peptide with a m/z value of 2513 was not observed with either enrichment approach used in this work. Comparison of Phosphopeptide Isolation Using Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns and Titanium Dioxide Tips from Synthetic Mixtures. To better evaluate the performance of immobilized metal affinityreversed phase microcolumns and titanium dioxide for the selective capture of phosphopeptides, moderately complex synthetic mixtures consisting of phosphorylated (β-casein and ovalbumin) and nonphosphorylated (BSA and β-lactoglobulin) protein digests were enriched. The selectivity and sensitivity of each approach was assessed by enriching mixtures of increasing nonphosphorylated protein digest content in the following ratios: 1:1:1:1, 1:1:20:20, and 1:1:50:50. Figure 3 depicts the MALDI mass spectra resulting from enrichment of each ratio using biphasic immobilized metal affinity-reversed phase microcolumns. Capture for the 1:1:1:1 mixture was successful for all six phosphopeptides detected in the enrichment of the single phosphoprotein digests of β-casein and ovalbumin without a substantial presence of nonphosphorylated peptides (Figure 3a). Table 2 lists the corresponding m/z values for each detected phosphopeptide.Increasing the amount of the two nonphosphorylated protein digests 10-fold each allowed for the detection of five phosphopeptides with minimal spectral contamination resulting from nonspecific binding (Figure 3b). Further increasing BSA and β-lactoglobulin amounts 50-fold each, four of the six phosphopeptides are detected as seen in the MALDI mass spectrum of Figure 3c. At this ratio, coenrichment of nonphosphorylated peptides begins to become significant. It must be noted, however, that enrichment of increasing ratios results in lower signal intensity of all phosphopeptides. In contrast, upon titanium dioxide enrichment of the 1:1: 1:1 mixture, only four phosphopeptides were detected (Figure 4a). Table 2 lists the corresponding m/z values for each detected phosphopeptide. At an increased ratio of 1:1:10:10 for enrichment, these phopshopeptides are still detected, albeit at a much lower signal intensity, but with a dominant occurrence of nonspecific binding as depicted in the MALDI mass spectrum of Figure 4b. At a mixture ratio of 1:1:50:50 only nonphosphorylated peptide signals are observed (Figure 4c). This may be due perhaps to the titanium dioxide surface binding sites being overwhelmed by nonphosphorylated peptides or due to ionization suppression. Thus, it appears that the biphasic immobilized metal affinity-reversed phase enrichment provides a more selective as well as sensitive method for phosphopeptide isolation compared to the titanium dioxide approach used in this work. Phosphopeptide Enrichment from β-Casein Monophosphopeptide Spiked Human Serum via Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns. The sensitivity of phosphopeptide isolation with the newly developed biphasic microcolumn approach for real, complex biological samples was determined by spiking various amounts of β-casein monophosphopeptide into a constant volume (and thus constant total protein content) of trypsin digested human serum. Phosphopeptide enriched samples were subsequently analyzed using LC-ESI-MS3 via neutral loss detection. Figure 5 depicts base peak chromatograms of the enriched digested human serum spiked with 0.2 to 20 pmol of β-casein monophosphopeptide. The doubly charged ion (m/z 1032) of the β-casein monophosphopeptide is represented by the most intense signal in the chromatogram at the 20 pmol spike level with a retention

technical notes

Enrichment of Phosphopeptides time of 52 min (Figure 5a). Reduction of the β-casein monophosphopeptide to 10 pmol still allows for its detection, although as a shoulder of a more intense peak with a m/z value of 517 (Figure 5b). Further reduction of β-casein monophosphopeptide amounts did not allow for the detection of the doubly charged ion (Figure 5c-e).

Conclusion The biphasic immobilized metal affinity-reversed phase microcolumn methodology presented in this work utilizing the Pro-Q Diamond resin allows for the selective enrichment of phosphopeptides from phosphoprotein digests. In comparison to a titanium dioxide approach, the biphasic microcolumn method was found to be more specific and sensitive for phosphopeptide capture from biological samples of higher complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of β-casein monophosphorylated peptide in 20 µL of a solution of human serum protein digest (from 200 µg total serum protein after digestion and desalting). The method is simple and fast as well as applicable to both MALDI and electrospray based mass spectrometry.

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