Mass Spectrometric Peptide Fingerprinting of Proteins after Western

The most popular supports for Western blot applications include NC and PVDF. ... Enhanced chemiluminescence reagents, Triton X-100, and Hyperfilm ECL ...
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Mass Spectrometric Peptide Fingerprinting of Proteins after Western Blotting on Polyvinylidene Fluoride and Enhanced Chemiluminescence Detection Ruth Menque Methogo, Genevie` ve Dufresne-Martin, Patrice Leclerc, Richard Leduc, and Klaus Klarskov* Department of Pharmacology, Faculty of Medicine, Universite´ de Sherbrooke, Sherbrooke, Quebec, Canada Received January 24, 2005

The combined use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry has become a powerful and widely used tool in proteome studies. Following separation by electrophoresis, proteins can be transferred to an inert support such as polyvinylidene fluoride (PVDF) or nitrocellulose (NC) for the visualization of individual or specific classes of proteins by immunochemical detection methods. We developed a method that allows the mass spectrometric analysis of peptides derived from proteins detected by Western blotting on PVDF. Proteolysis buffer containing either dimethyl formamide (DMF) or Triton X-100 to recover peptides amenable to mass spectrometry was investigated. Although either one can be used, the buffer containing DMF required less sample handling prior to mass spectrometry. The approach was tested using commercially available proteins and serinephosphorylated proteins from an HEK-293 nuclear extract. Keywords: peptide mapping • India ink staining • mass spectrometry • semidry electroblotting • immunostaining • protein phosphorylation

Introduction The high throughput DNA sequencing of genomes in the past few decades has paved the way for the development of tools for the detailed, differential analysis of the cellular proteome. Especially, approaches based on sodium dodecyl sulfate polyacrylamide electrophoresis and mass spectrometry have gained wide popularity. They are now being used in the field of proteomics to study a variety of disease processes.1-4 The two main methods of choice for obtaining peptides from electrophoretically separated proteins of interest that are amenable for mass spectrometry are in-gel and on-membrane proteolytic digestion or chemical cleavage. Both methods have inherent strengths and weaknesses. In-gel proteolysis requires fewer steps from initial separation through to mass spectrometryready samples. However, this approach has a number of major limitations, including extensive cleavage time (typically overnight), reduced proteolytic efficiency resulting in incomplete cleavage, the need for relatively large sample volumes, and the difficulty in combining the method with immunochemical staining approaches. On the other hand, on-membrane proteolytic cleavage of proteins of interest requires an additional blotting step after the electrophoretic separation but shorter cleavage times can be used and, more importantly, it can be combined with immunochemical staining. Immunochemical detection in combination with one or two-dimensional electrophoresis is widely used in proteomics for the differential * To whom correspondence should be addressed. Mass Spectrometry Laboratory, Department of Pharmacology, Faculty of Medicine, Universite´ de Sherbrooke, 3001-12e, Avenue Nord, Sherbrooke, Quebec, Canada, J1H 5N4. Tel.: (819) 820-6868, ext. 1-5490. Fax: (819) 564-5400, E-mail: [email protected].

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visualization of subsets of proteins containing common markers, e.g., phosphorylation or other post-translational modifications.5,6 However, mass spectrometric analysis and identification of immunochemically detected proteins requires running two parallel gels, one for Western blotting and one for preparing peptides from the corresponding stained gel or membrane.7,8 Nonetheless, it is frequently very difficult to assign the bands on a Western blot to those on a stained gel. The most popular supports for Western blot applications include NC and PVDF. Compared to NC, the chemical and physical stability together with high protein binding capacity of PVDF makes it the preferred choice in many applications. The purpose of our study was thus to develop a method to analyze, by mass spectrometry, peptides derived from proteins detected by Western blotting on PVDF membranes. Even though accurate peptide mass determination by MALDI-MS is a relatively reliable method for the identification of unknown proteins, the selectivity and degree of confidence of the identification can be considerably increased when tandem mass spectrometry data are included. This is currently the accepted standard for peptide identification.9 We thus also looked at whether the Western blot approach was compatible with electrospray ionization time-of-flight tandem mass spectrometry (ESI-MS/ MS) coupled on-line with high performance nano liquid chromatography (nLC). Recovering proteolytically derived peptides from extremely hydrophobic membranes such as PVDF is, from a mass spectrometric point of view, a challenging task requiring the use of high concentrations of either organic acids, organic solvents, or nonpolar detergents.10 We evaluated two ammonium bicarbonate buffers, one containing dimethyl formamide (DMF) and the other, Triton X-100, for the concur10.1021/pr050014+ CCC: $30.25

 2005 American Chemical Society

MS Peptide Fingerprinting of Proteins

rent extraction and tryptic cleavage on PVDF membranes. For the proteins tested in this study, both buffers resulted in similar peptide recoveries and could be used to obtain peptides suitable for mass spectrometry allowing potential identification of proteins detected on Western blots using enhanced chemiluminescence (ECL). The obvious advantage of this approach is a reduction in the time necessary for sample preparation, since only a single electrophoretic separation is required. We applied the approach to the 2-D Western blot analysis of phosphorylated nuclear proteins obtained from an HEK-293 cell line.

Materials and Methods Materials. E.coli β-galactosidase, bovine serum albumin (BSA), human lactoferrin, human fibronectin (70 kDa truncated form), chicken ovalbumin, rabbit skeletal actin, and all rabbit polyclonal primary anti-sera were purchased from Sigma (St. Louis, MO). PVDF membranes, ZipTip C18, and ZipTip SCX were from Millipore (Bedford, MA). Trypsin was from Promega (Madison, WI). Fount India ink (Pellikan, Pembroke, MA) was purchased in a local art store. Criterion gel cassettes (1 mm thick) and all electrophoresis reagents were from Bio-Rad Laboratories (Mississuaga, ON). Anti-phosphoserine monoclonal antibody was from Research Diagnostics (Flanders, NJ). 5-methoxysalicylic acid and R-cyano-4-hydroxycinnamic acid were from Aldrich (Milwaukee, WI). Enhanced chemiluminescence reagents, Triton X-100, and Hyperfilm ECL were from Amersham Pharmacia Biotech (Baie d’Urfe´, QC). All other reagents and solvents were of the highest grade. SDS-PAGE, Staining, and Electroblotting. Stock solutions of proteins (β-galactosidase, BSA, lactoferrin, fibronectin, ovalbumin, and actin) were prepared in water at a concentration of 100 pmoles/µL. Prior to electrophoresis, they were diluted in 2× Laemmli’s sample buffer to appropriate concentrations in order to apply 5 µL onto the gel. The proteins were separated on 7% SDS polyacrylamide minigels using the Criterion gel system and a constant current of 50 mA/gel. After electrophoresis, the proteins were either stained with SYPRO orange11 or transferred to PVDF membranes (pre-wetted in methanol and blot buffer) by semidry blotting using a Trans-Blot SD semi-dry transfer cell (Bio-Rad) for 55 min at 0.8 mA/cm2 as described elsewhere.12 After electroblotting, the PVDF membranes were rinsed for 5 min in 20% (v/v) methanol and blocked with 0.2% (v/v) Tween 20 in 0.1% (v/v) acetic acid for 10 min. The proteins were stained in PBS containing 0.05% (v/ v) Tween 20, 1% (v/v) acetic acid, and 0.1% (v/v) fount India ink until visible bands appeared (30 min to 3 h). HEK-293 Cell Sample Preparation and 2D Electrophoresis. HEK-293 cells stably expressing the hAT receptor were seeded into 10 Petri dishes (15 cm I. D.) and grown to confluence. They were then washed repetitively 3x with PBS and harvested in 10 mM Hepes buffer pH 8.0 containing 10 mM KCl, 1.5 mM MgCl2, and 1 mM dithiothreitol. A nuclear extract was prepared using a previously described approach.13 Protein concentrations were determined using the BCA protein assay (Pierce) after diluting the sample in 1% SDS. IEF was performed using ReadyStrips (pH 3-10L) and the PROTEAN IEF Cell (Bio-Rad). Homogenized samples were diluted to a protein concentration of 2.4 µg/µL in IEF sample buffer (8 M urea, 2% CHAPS, 65 mM dithiothreitol, 1:50 volume of Biolyte pH 3-10, and a trace of bromophenol blue). A total of 185 µL of sample buffer containing 450 µg of protein was used to rehydrate the strip following the manufacturer’s protocol. The proteins were

research articles focused at 20 °C using the following voltage gradient: 0-250 V over 20 min, 250-8000 V over 2.5 h, and 8000 V for 2.5 h. Prior to electrophoresis in the second dimension on 10% gels, the proteins were reduced in equilibration buffer (50 mM TrisHCl pH 8.8 containing 6 M urea, 30% glycerol and 2% SDS, 2% DTT) for 10 min followed by carbamidomethylation in the same buffer containing 25 mg/mL iodoacetamide for 10 min in the dark. Electroblotting was performed as described above. Immunochemical Detection. After electroblotting, blocking, and India ink staining, the PVDF membrane was rinsed in water and wash buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.6, 0.05% Tween 20) followed by incubation with the primary antibody (dil. 100 000×) in wash buffer. The membrane was rinsed after 2 h (4 × 5 min) with wash buffer and incubated with monkey horseradish peroxidase-conjugated anti-rabbit IgG antibody for 1 h. Phosphorylated proteins separated by 2D SDS-PAGE were detected on blots using anti-phosphoserine monoclonal antibodies (diluted 1000×) and secondary horseradish peroxidaseconjugated antibody (diluted 100 000×). Spots of interest were excised and, if not used immediately, stored in Eppendorf tubes at -20 °C. Proteolytic Cleavage, Sample Preparation, and Mass Spectrometry. In-gel trypsin digestions were performed, with few modifications, as described elsewhere.12 Briefly, protein spots excised from the gels were equilibrated in 400 µL of 100 mM NH4HCO3 for 15 min at room temperature followed by dehydration in acetonitrile. The proteins were reduced by adding 50 µL of 100 mM NH4HCO3 containing 5 mg/mL DTT to the dehydrated gel spots. After 45 min at 37 °C, the buffer was replaced with 200 mM ammonium bicarbonate buffer containing 25 mg/mL iodoacetamide, and the carbamethylation was allowed to proceed for 30 min in the dark at room temperature. Excess reagent was replaced with 400 µL of 100 mM NH4HCO3/ 50% acetonitrile and the gel slices were incubated for 30 min. The solvent was removed and the gel slices were lyophilized for 2 min. The gel slices were rehydrated in 25 µL of a solution containing trypsin (50 ng/µL) dissolved in 50 mM NH4HCO3/ 40% DMF for 15 min at room temperature. The buffer, not absorbed by the gel, was then removed and 50 µL of 50 mM NH4HCO3 was added. The trypsin digestion was performed overnight at 37 °C. The peptides were concentrated after the addition of 5 µL 10% formic acid on a ZipTip C18 following the manufacture’s instructions and loaded onto the MALDI-target as described below. Proteins intended for on-membrane digestion, were excised and reduced in 10 µL of 100 mM NH4HCO3 containing 5 mg/ mL DTT for 45 min at 37 °C. The carbamidomethylation was performed in the dark in 20 µL of 200 mM NH4HCO3 containing 25 µg/µL of iodoacetamide. In some cases, excess antibody was removed prior to trypsin digestion by washing the PVDF membranes in 20% DMF/1% formic acid. The removal of the primary antibody was verified in a separate experiment, by incubating the membrane with the secondary antibody followed by ECL. Membrane pieces containing the proteins of interest were rinsed four times in 400 mL 50 mM ammonium bicarbonate containing 10% acetonitrile. Digestion on the membrane was performed at 37 °C for 3.5 h in 8 µL of 50 mM NH4HCO3 containing 20 ng/µL of trypsin and either 1% Triton X-100 or 50% DMF. The samples were centrifuged every 30 min to prevent the membranes pieces from drying out. The samples were sonicated for 2 min and 30 µL 5% formic acid was added to the samples digested in NH4CO3/DMF, followed by immediate concentration of the peptides on a ZipTip C18 according to Journal of Proteome Research • Vol. 4, No. 6, 2005 2217

research articles the manufacturer’s instructions. If the sample was intended for nLC-ESI-MS/MS, 1 µL of 5 M urea solution was added before lyophilization for 30 min at room temperature using an Eppendorf Vacufuge Concentrator and a maximum vacuum specified to be better than 20 mbar. Samples digested in the buffer containing 1% Triton X-100 were diluted with 40 µL 0.05% formic acid followed by concentration on a ZipTip SCX for cation exchange concentration of the peptides. The detergent was removed with 3 × 10 µL washes in 35% acetonitrile containing 0.05% formic acid and the peptides were eluted with 5 µL 1% ammonium hydroxide solution containing 30% methanol. The solution was acidified by the addition of 5 µL 2% formic acid. The peptides were concentrated using a ZipTip C18 column according to the manufacturer’s instructions and eluted with 1.5 µL MALDI-MS matrix solution (12 mg/mL alpha-cyano-4-hydroxy-cinnamic acid in 45% acetonitrile). A 1-µL aliquot was applied to the MALDI-MS target prepared with a precrystallized ultrathin layer of 5-methoxysalicylic acid using a protocol similar to the one described by Cadene et al.14 On-membrane digested samples intended for nLC-ESI-MS/ MS analysis were dissolved in 14 µL of solvent A (1% acetonitrile/1% 2-propanol/0.2% formic acid in water) prior to injection of 10 µl. MALDI-MS mass spectra were recorded on a TofSpec 2E (Waters/Micromass Corp., UK) using the delayed extraction and reflector mode. Acceleration and pulse voltages were set at 20 000 and 2300 V, respectively, and the low-mass gate was 500. External calibration was performed with a mixture of angiotensin I and reduced carbamidomethylated porcine insulin B-chain. nLC-ESI-MS/MS analyses were performed on a Q-TOF-2 (Waters/Micromass Corp.) coupled on-line to a CapLC pump (Waters Corp.) equipped with three separate syringe pump modules, an auto injector, a 10 port valve and a 250 µm I.D × 1 mm precolumn. Separations were performed on a 7 cm × 75 µm I.D. capillary column. Both columns were packed with Microsorb C18 (Varian Inc., Mississuaga, ON) reversed-phase material. Peptides were eluted at a flow rate of 0.25 µL/min with a linear gradient of solvent B (80% acetonitrile containing 10% 2-propanol and 0.2% formic acid) in solvent A: from 0 to 60% B over 40 min, to 90% B over 7 min, and 10% B over 8 min. Survey scans were acquired at 1.9 s/scan. Depending on the charge state and intensity, parent ions were automatically selected for tandem mass spectrometry and acquired at 1 s/scan with a pre-set maximum of 4 scans per selected parent ion. The Q-TOF mass spectrometer was calibrated by infusing a solution of 0.2 µg/µL of NaI dissolved in 50% 2-propanol (0.2 µg/µL) or 1 pmol/µL of Glu-fibrinopeptide B dissolved in 30% acetonitrile containing 0.2% formic acid. Parameters for Database Searching. Peptide mass searches were performed with the Protein Prospector search engine (http://prospector.ucsf.edu) and the MASCOT algorithm (http:// www.matrixscience.com). The latest versions of the NCBI and Swiss-Prot protein databases were used. Peak lists were prepared from either MaxEnt 3 (part of the MassLynx 3.5 software) transformed MALDI-MS spectra or smoothed, backgroundsubtracted, and centroided MS/MS spectra as input data. When using the accurate masses from the MALDI-MS spectra as input data, a mass accuracy greater than 100 ppm, an allowance of two incomplete proteolytic cleavage sites, cysteine carbamidomethylation, methionine oxidation, and selection of the species was specified in the search program. The MASCOT software default settings were used for protein identification with MS/MS-derived data. 2218

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Methogo et al. Table 1. Comparative MALDI-MS Peptide Mass Fingerprinting Using Tryptic Peptides Obtained after 1D PAGE in-gelb

on-membranec

proteina

40% DMF (no. pept/%covd)

1% Triton X-100 (no. pept/%covd)

50% DMF (no. pept/%covd)

BSA Ovalbumin Lactoferrin Actin galactosidase fibronectin

20/44 11/39 24/44 15/40 27/30 23/46

25/44 9/35 19/29 10/36 32/29 12/29

22/42 11/39 22/36 12/33 31/34 13/28

a The sequences used for calculation of sequence coverage are the following: BSA, AAA51411; ovalbumin, OACH; Lactoferrin, 1LFH; Actin, ATRB; β-galactosidase, CAA04788; Fibronectin, CAD59389. An estimated 5 pmol of each sample was loaded onto the gel. b Proteins were stained with Sypro orange (see Materials and Methods). c Tryptic digestion of India ink stained proteins was performed in 50 mM ammonium bicarbonate buffer containing either 1% Triton X-100 or 50% DMF. d Number of peptides assigned/% sequence coverage as deduced from MASCOT. The data are representative of three independent analyses.

Results and Discussion Peptide Mass Fingerprinting of India Ink-Stained Proteins Immobilized on PVDF. Taking into account the highly mechanically solid nature, chemical resistance, and hydrophobic properties of PVDF that provide for strong protein retention, we first evaluated two different solvent compositions to recover proteolytically derived peptides for MALDI-MS/MS. The use of volatile hydrophobic buffer mixtures containing DMF, methanol, or acetonitrile was recently proposed to improve proteolytic cleavage in solution,15 on PVDF,16 and for in-gel digestion.17 Detergents such as zwittergent 3-16 and Triton X-100 improve peptide recovery from on-membrane digestions18,19 but require removal from the peptides prior to mass spectrometry. While small amounts of zwittergent can be retained on a ZipTip C18, high amounts rapidly saturate the reverse phase material and affect retention of the peptides. Unlike Triton X-100 and due to the zwitterionic nature, zwittergent cannot be removed using a ZipTip SCX. On the basis of these considerations, we chose to determine whether digestion buffers containing 50% DMF or 1% Triton X-100 in 50 mM ammonium bicarbonate were compatible with downstream MALDI-MS and whether the data was comparable to that obtained after in-gel digestion. Hence, six commercially available proteins were separated by 1-D SDS-PAGE. After electroblotting onto PVDF and blocking with 0.2% Tween 20, the proteins were stained with India ink because it is compatible with immunodetection techniques.20 The proteins on excised spots were reduced, carbamidomethylated, and digested with trypsin. For the digestions in buffer containing 1% Triton X-100, we used two purification steps to remove the detergent and salts before the MS analysis. The first consisted of trapping the peptides on a ZipTip SCX pipet tip and eluting the detergent with 35% aqueous acetonitrile containing 0.05% formic acid. The peptides were then eluted and concentrated using a ZipTip C18 pipet tip. Proteins digested in buffer containing DMF were diluted with aqueous formic acid and concentrated using a ZipTip C18 pipet tip before the MALDIMS analysis. The sequence coverage data obtained using the observed accurate masses as input data and the search engine MASCOT to explore the most recently updated NCBI protein database are summarized in Table 1. Except for ovalbumin, β-galactosidase, and BSA, in-gel proteolysis resulted in a minor improvement in sequence coverage compared to the corresponding on-membrane digestions. It is important to mention

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Table 2. Comparative MALDI-MS PMF after Western Blotting on PVDF and On-Membrane Trypsin Digestion 1% Triton X-100a

50% DMFa

proteinb

5 pmol ECL (no. pept/%covd)

5 pmol ECL-Abc (no. pept/%cov)

1 pmol ECL (no. pept/%cov)

5 pmol ECL (no. pept/%cov)

5 pmol ECL-Abc (no. pept/%cov)

ECL (no. pept/%cov)

BSA ovalbumin lactoferrin actin galactosidase fibronectin

24/45 9/34 18/30 10/36 33/34 16/28

15/33 9/29 19/29 8/26 30/31 11/21

12/23 9/34 15/24 10/35 24/24 10/18

17/40 11/39 23/40 12/40 31/37 14/29

12/26 10/36 15/26 9/33 25/32 14/29

10/20 11/39 20/29 12/36 26/25 13/21

a Tryptic digestion of India ink stained proteins was performed in 50 mM ammonium bicarbonate buffer containing either 1% Triton X-100 or 50% DMF. The sequences used to calculate sequence coverage are the following: BSA, AAA51411; Ovalbumin, OACH; Lactoferrin, 1LFH; Actin, ATRB; β-galactosidase, CAA04788; Fibronectin, CAD59389. c Excess antibodies was removed by washing the membrane with 20% DMF containing 1% formic acid d Number of peptides assigned/% sequence coverage as obtained from MASCOT. The data are representative of three independent analyses. b

that the variability in the determined sequence coverage as determined from 3 to 5 repetitive analyses was less than 5%. On-membrane digestions in the presence of Triton X-100 or DMF gave comparable results with regard to the number of peptides observed and sequence coverage. Ovalbumin is difficult to digest unless extensively denatured and even resists endo Lys-C proteolysis on PVDF in the presence of 80% acetonitrile.16 Interestingly, ovalbumin was efficiently denatured in a buffer containing either DMF or Triton X-100, allowing the recovery of an adequate amount of peptides to unambiguously identify the protein (Table 1). Western Blot-MALDI-MS. The purpose of the present study was to extend our previous work dealing with protein identification on nitrocellulose membranes21 to include mass spectrometric analysis of peptides and protein identification after Western blot on PVDF membranes. Following electrophoresis and electroblotting, six proteins were probed with the appropriate antisera and visualized by ECL. With conventional Western blots, blocking the membrane with proteins such as BSA or casein prevents nonspecific adsorption of antibodies and extensive background staining. However, we anticipated that excess proteins on the membrane might significantly interfere with the mass spectrometric analysis and potentially compromise the identification of picomol and sub-picomol quantities of unknown proteins. In addition, using Tween 20 in the blocking solution rather than a protein increases the sensitivity of the immuno detection and prevents nonspecific binding of antibody.21 Therefore, a more diluted antibody preparation had to be used to obtain a similar immunostaining versus background intensity ratio as that obtained when the membrane is blocked with a protein-containing solution (data not shown). The PVDF membranes were thus blocked with Tween 20 prior to India ink staining and incubation with the antibodies. While total protein staining with India ink can be avoided, it facilitates considerably the location of spots of interest. The major disadvantage of India ink staining is the reduced contrast following immunostaining due to a partial loss of the colloidal India ink stain in the washing solutions. Nevertheless, we anticipate that other reversible staining methods based on metal chelates,22 colloidal silver,23-25 or SYPRO Rose Plus26 could replace the India ink staining, although they should be performed after the immunostaining, due to the lack of compatibility. The results of comparative peptide mass fingerprinting (PMF) by MALDI-MS of the six proteins immunochemically detected on PVDF after visualization with ECL are summarized in Table 2. As expected, there were no major differences in the number of peptides detected versus the sequence coverage with the two buffers using a 5

pmol sample load. However, decreasing the amount of sample loaded on the gel to 1 pmol decreased the number of peptides and sequence coverage (Table 2). Representative spectra of the six proteins digested in buffer containing DMF are shown in Figure 1A-F. Marked differences in signal intensities can be seen, which may be due to variations in transfer efficiency during the blotting process and/or recovery of the proteins from the PVDF membrane. However, in all cases, the number of peptides detected was sufficient to tentatively identify the proteins. Surprisingly few peaks were observed in the spectrum of a piece of blank PVDF membrane handled in the same way as the sample spots (Figure 1G). In addition, the removal of excess of antibodies prior to the on-membrane digestion did not result in an improved PMF due to a reduced number of peptides detected, except in the case of lactoferrin, when digested in the presence of Triton X-100 (Table 2). A plausible explanation for this observation could be a potential loss of protein from the membrane during the washing step, which would result in a reduced amount of protein available for trypsin digestion and consequently a lower quantity of peptide for the MS analysis. The sensitivity of the combined approach was estimated at approximately 0.5 pmol for ovalbumin (Figure 2). When the buffer containing DMF was used, we detected 11 peptides that covered 37% of the sequence. However, with the same amount of sample, we were unable to detect any peptides when the buffer containing Triton X-100 was used. Peptides were likely partially lost during the two purification steps required for the removal of the Triton X-100 prior to the MALDI-MS analysis, reducing the sensitivity. Western Blotting and Compatibility with nLC-MS. To evaluate whether the Western blot approach was compatible with nLC-ESI-MS/MS, 0.5 pmol of each protein was loaded onto a gel and after 1D SDS-PAGE, electroblotted onto a PVDF membrane. Following blocking with Tween 20, India ink staining, and immunodetection, the proteins were reduced, alkylated, and digested with trypsin. Since additional steps are required to remove Triton X-100, which interferes with nLCESI-MS/MS analysis, we decided to focus on the results obtained using buffer containing DMF, which can be removed by simple lyophilization. An example of an nLC-MS/MS analysis of peptides derived from an on-membrane digestion of lactoferrin is shown in Figure 3A. A representative tandem mass spectrum of the doubly charged ion (m/z 902.9) that could be assigned to the tryptic peptide Gly387-Lys404 is shown in Figure 3B. A total of 13 automatically selected peptides resulted in useful tandem mass spectrometry data that could be unambiguously matched to the sequence of human lactoferrin with a MASCOT probability score of 720. To use a low threshold Journal of Proteome Research • Vol. 4, No. 6, 2005 2219

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Figure 1. Proteins (1 pmol) separated by 1D PAGE on 7% acrylamide gels were transferred to a PVDF membrane by electroblotting and stained with India ink. Following probing with the respective antibodies and visualization with ECL, protein spots were excised, reduced, alkylated, and digested with trypsin in proteolysis buffer containing DMF as described in the Materials and Methods section. The peptides were analyzed by MALDI-MS. (A) BSA, (B) ovalbumine, (C) lactoferrin, (D) actin, (E) β-Galactosidase, (F) fibronectin, 70 kDa truncated form, and (G) blank piece of membrane without protein treated in the same way as the samples. For comparison purposes, sixty laser shots were combined per spectrum and the intensity (Y-scale) was set to 1100 counts in all spectra. Only the mass values used in PMF are labeled.

value setting during automatic ion selection and reduce the selection of potential noise and peaks due to impurities, only 2220

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doubly and triply charged molecular ions above a predefined value were chosen for the tandem mass spectrometry. Singly

MS Peptide Fingerprinting of Proteins

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Figure 2. MALDI-MS analysis of tryptic peptides derived from on-membrane digestion of ovalbumine (as described in Figure 1) after blocking the PVDF with Tween 20, India ink staining, and immunochemical detection by ECL (insert). An estimated amount of 500 femtomoles was loaded onto the gel. Only the mass values used for PMF are labeled. Sixty laser shots were combined per spectrum and the intensity (Y-scale) was set to 1100 counts.

Figure 3. nLC-ESI-MS/MS analysis of tryptic peptides derived from on-membrane digestion of lactoferrin (500 femtomoles were loaded onto the gel) after immunochemical detection. The proteolysis was performed in DMF buffer. (A) Base peak ion chromatogram of the survey scans acquired and (B) Tandem mass spectrometry of the doubly charged peptide ion (m/z 903). The retention time is shown with an arrow in (A). Four 1 s/scan spectra were combined.

charged peptides and peptides with a charge state higher than 3, were not submitted to fragmentation during these analyses. Since polymer ions (Tween 20) elute relatively late in the gradient and are detected as singly charged ions, they do not interfere with the fragmentation of multiply charged peptide ions. The increased retention time of the detergent could indicate that it is not eluted from the ZipTip at the conditions used to elute the peptides, explaining the absence from the

MALDI spectra shown in Figure 1. Analysis by nLC-ESI-MS/ MS of 0.5 pmol BSA, ovalbumin, actin, and β-galactosidase after ECL detection resulted in the selection of 6, 5, 6, and 8 ions, respectively, that provided adequate sequence information that could be matched to the respective protein sequences. In the case of fibronectin, only two peptide ions of sufficient intensity gave spectra that could be successfully assigned to the amino acid sequence. We are presently uncertain as to the reason for Journal of Proteome Research • Vol. 4, No. 6, 2005 2221

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Table 3. Serine Phosphorylated Proteins from HEK 293 Nuclear Cell Extracts Identified after Detection by 2D Western Blotting spota

assigned protein

pIb calc.

Mr mass |(Da)

1

Translation elongation factor eEF-1 delta chain Nucleophosmin Sequence 23 from Patent WO0159155 Translation initiation factor 4AII Alpha enolase Heterogeneous nuclear ribonucleoprotein B1 Splicing factor Arg/Ser rich PDZ and LIM dom. 1 (elfin) Heterogeneous nuclear ribonucleoprotein 2H9 Heterogeneous nuclear ribonucleoprotein 2H9 Glyceraldehyde-3phosphate dehydrogenase Heterogeneous nuclear ribonucleoprotein B1

4.95

31.202

4.64 5.39

2

3 4 5 6 7 8

MASCOT score

no. of peptidesc

no. of P-sitesd

S34626

110

2

7

32.555 47.43

A32915 CAC69372

43 170

2 7

17 8

5.33

46.365

BAA06336

47

2

5

6.99 8.97

47.008 37.407

ENOA B34504

1082 115

18 5

8 12

5.61 6.56 6.37

31.979 36.049 36.903

B40040 AAH00915 AAF68843

33 80 61

1 2 2

5 9 8

6.37

36.903

AAF68843

66

2

8

8.57

36.03

DEHUG3

74

2

11

8.97

37.407

B34504

34

2

12

AC no.

a Spots were obtained from the corresponding India ink-stained PVDF membrane used for ECL detection, shown in Figure 4. b The calculated molecular masses and pI were obtained from MASCOT. c Number of peptides from the nLC-ESI-MS/MS analysis that could successfully be assigned to the protein amino acid sequence. d The numbers of predicted serine phosphorylation sites were determined using the CBS prediction server http://www.cbs.dtu.dk/ services/

this relatively low peptide recovery. With 0.5 pmol of sample, fibronectin is barely visible after immunostaining, and since the protein was not stained again with India ink before excising the spot, some sample loss at this level could not be ruled out. When 0.25 pmol was loaded onto the gel, we were unable to identify fibronectin. However, with the proteins that could be seen with India ink after the immunodetection step, including β-galactosidase, BSA, and actin, summed MASCOT probability scores well above the level of significance (>53) were obtained from the 8 (score: 280), 7 (score: 540), and 3 (score: 95), tandem mass spectra of selected peptide ions, respectively. Identification of Immunochemically Detected Serine Phosphorylated Proteins. There is growing interest in the analysis of discrete subgroups of proteins. A first step toward such analyses is the separation and differential detection of the subclasses of proteins of interest in the entire proteome. Protein phosphorylation is one of the most common post-translational modifications that occurs in nature and has attracted special interest because of its importance in numerous cellular processes. While an elegant multiplexed fluorescence method has recently been developed to specifically detect phosphorylated proteins separated by gel electrophoresis and electroblotted onto PVDF or nitrocellulose,27-29 the use of Western blotting remains one of the most widespread and versatile methods for detecting these modifications. To determine whether our approach could be used to analyze a subclass of proteins among a complex protein mixture, we applied it to the detection of serine phosphorylated proteins derived from a nuclear HEK-293 cell extract. The proteins were separated by 2-D SDS-PAGE and electroblotted onto PVDF. After blocking the membrane with Tween 20 and staining with India ink, phosphorylated proteins were probed with a polyclonal antibody used to detect serine phosphorylation and visualized by ECL as shown in Figure 4. Eight of the most intensely India ink-stained spots were excised after ECL detection and reduced, alkylated with iodoacetamide, and digested with trypsin. With a few exceptions, preliminary analyses of the tryptic peptides with MALDI-MS were unsuccessful (data not shown). This may 2222

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Figure 4. Detection of serine phosphorylated nuclear proteins after 2-D immunoblotting and ECL detection on PVDF. Nuclear proteins (450 µg total protein) isolated from HEK-293 cells were separated in the first dimension by immobilized pH gradient isoelectric focusing (IPG pH 3-10, 11 cm long) and in the second dimension using a 10% SDS-PAGE gel. After separation, the proteins were transferred to a PVDF membrane by semidry electroblotting. After blocking with Tween 20, they were stained with India ink prior to probing with an anti-phosphoserine monoclonal antibody (diluted 1000×). Phosphorylated proteins were visualized after incubation with horseradish peroxidaseconjugated anti-IgG secondary antibody (diluted 100.000x). The numbered immunoreactive spots were excised from the corresponding India ink-stained membrane, digested with trypsin, and analyzed by nLC-ESI-MS/MS.

have been due to the limited amount of sample that could be applied to the minigels employed throughout this study. Instead, we then used nLC-ESI-MS/MS to analyze the tryptic peptides. One advantage of this technique is that samples do not require preconcentration on ZipTips, thus reducing handling and the risk of sample loss. The results are summarized

research articles

MS Peptide Fingerprinting of Proteins

in Table 3. As shown, half the selected protein spots appeared to be heterogeneous, precluding unambiguous identification of the phosphorylated protein(s). According to the CBS prediction server,30 all the proteins identified in this study can be phosphorylated confirming the specificity of the Western blot approach. However, this emphasizes the need for homogeneous protein spots, except when modified peptides can be detected and assigned to a protein. Two protein assignments had scores that were slightly lower than the level of significance required by the MASCOT search program. On the basis of the MS/MS data from one peptide, one protein was identified as Arg/Ser-rich splicing factor. In addition, the PMFs of five other peptides that were detected in the survey scans from the LCMS/MS analysis and that were not selected for tandem MS could be assigned to the sequence. Moreover, the calculated pI (5.61) and Mr mass (31.979 Da) corresponded to the roughly estimated values based on the 2-D gel pattern, considering the values for other protein spots that had already been identified. The identification of heterogeneous nuclear protein B1 in spot 4 could be due to a variant being more heavily phosphorylated than the isomer identified in spot 8, causing a shift toward a lower pI. Nevertheless, the protein assignments for spots 5 and 8 require further validation using an independent approach such as immunodetection techniques with specific antibodies. We focused on analyzing the most abundant protein spots, based on the intensity of the India ink staining and taking into account the limited amount of sample that could be loaded (300-500 µg/gel) versus the sample complexity and high sensitivity of immunostaining. The analysis of less abundant proteins would require the pooling of spots from several 2D blots. Narrow range IEF gels could be employed alternately or in combination, allowing a larger total sample load to be used.

Conclusion We demonstrate the feasibility of performing PMF using MALDI-MS and nLC-ESI-MS/MS analyses of peptides derived from India ink-stained proteins detected immunochemically on PVDF. The method takes advantage of the chemical resistance of PVDF membranes, which allows the use of a high concentration of organic solvent in the proteolysis buffer, eliminating the use of detergents, and simplifying the peptide purification step. While the use of Triton X-100 in the proteolysis buffer requires an additional purification step to remove the detergent prior to mass spectrometry, DMF can be removed by lyophilization. Furthermore, proteolysis on PVDF in the presence of an increased concentration of DMF could be an advantage for the digestion of certain proteins. For example, digestion of 0.5 pmol ovalbumin on PVDF, in the presence of 50% DMF, resulted in an improved peptide recovery and sequence coverage, compared to the same analysis on NC in the presence of zwittergent 3-16.21 With half of the sample loaded on the gel, 11 peptides were observed after proteolysis on PVDF, whereas 7 peptides were obtained on NC, representing 37 and 25% of the sequence, respectively. This approach also eliminates the need for the often-ambiguous comparison and, consequently, difficult correlation of protein spots detected immunochemically with those on a parallel stained gel or blot. Moreover, less time is required per analysis since only one separation step is needed. Although we focused on ECL detection, other principles of Western blot detection such as fluorescence and alkaline phosphatase can likely also be applied, as long the reagents do not cause

unknown covalent protein modification. However, using Tween 20 to block the membrane may cause higher background fluorescence and may have to be replaced by another polymer. We did not specifically look for tryptic peptides from the antibodies, and thus, their presence cannot be ruled out. However, as previously indicated, the amount is likely in the low femtomol range, and it does not appear to interfere with protein identification.21 The method is not restricted to the identification of phosphorylated proteins but should be useful for the analysis of a variety of subclasses of proteins with common post-translational modifications for which antibodies can be produced or purchased commercially as well as for the analysis of other subclasses of proteins that can be visualized selectively by other means.31 In principle, the major limitations include the high sensitivity of immunostaining versus sample amount required for mass spectrometry and the requirement for homogeneous protein spots. To circumvent these limitations, a combination of pre-fractionation, reducing the complexity of the protein mixture prior to electrophoresis, and conditions to accommodate higher sample amounts and to increase the resolution, are expected to be of importance.

Acknowledgment. Funding for this study came from Universite´ de Sherbrooke, the Fonds de La Recherche Sante´ du Que´bec (FRSQ), and the National Science and Engineering Research Council (NSERC). We thank Louise Barrette for her valuable help in preparing the manuscript. References (1) Lage, H. Pathol. Res. Pract. 2004, 200, 105-117. (2) Bukowska, A.; Lendeckel, U.; Kahne, T.; Goette, A. Pathol. Res. Pract. 2004, 200, 135-145. (3) Zhan, X.; Desiderio, D. M. Mass Spectrom. Rev. 2004. (4) Chambers, G.; Lawrie, L.; Cash, P.; Murray, G. I. J. Pathol. 2000, 192, 280-288. (5) Aulak, K. S.; Koeck, T.; Crabb, J. W.; Stuehr, D. J. Methods Mol. Biol. 2004, 279, 151-165. (6) Yuan, X.; Desiderio, D. M. J. Proteome Res. 2003, 2, 476-487. (7) Lock, R. A.; Coombs, G. W.; McWilliams, T. M.; Pearman, J. W.; Grubb, W. B.; Melrose, G. J.; Forbes, G. M. Helicobacter 2002, 7, 175-182. (8) Kaufmann, H.; Bailey, J. E.; Fussenegger, M. Proteomics 2001, 1, 194-199. (9) Carr, S.; Aebersold, R.; Baldwin, M.; Burlingame, A.; Clauser, K.; Nesvizhskii, A. Mol. Cell Proteomics 2004, 3, 531-533. (10) Jorgensen, C. S.; Jagd, M.; Sorensen, B. K.; McGuire, J.; Barkholt, V.; Hojrup, P.; Houen, G. Anal. Biochem. 2004, 330, 87-97. (11) Lauber, W. M.; Carroll, J. A.; Dufield, D. R.; Kiesel, J. R.; Radabaugh, M. R.; Malone, J. P. Electrophoresis 2001, 22, 906918. (12) Klarskov, K.; Naylor, S. Rapid Commun. Mass. Spectrom. 2002, 16, 35-42. (13) Dignam, J. D.; Lebovitz, R. M.; Roeder, R. G. Nucleic Acids Res. 1983, 11, 1475-1489. (14) Cadene, M.; Chait, B. T. Anal. Chem. 2000, 72, 5655-5658. (15) Russell, W. K.; Park, Z. Y.; Russell, D. H. Anal. Chem. 2001, 73, 2682-2685. (16) Bunai, K.; Nozaki, M.; Hamano, M.; Ogane, S.; Inoue, T.; Nemoto, T.; Nakanishi, H.; Yamane, K. Proteomics 2003, 3, 1738-1749. (17) Soskic, V.; Godovac-Zimmermann, J. Proteomics 2001, 1, 13642367. (18) Fernandez, J.; DeMott, M.; Atherton, D.; Mische, S. M. Anal. Biochem. 1992, 201, 255-264. (19) Lui, M.; Tempst, P.; Erdjument-Bromage, H. Anal. Biochem. 1996, 241, 156-166. (20) Eynard, L.; Lauriere, M. Electrophoresis 1998, 19, 1394-1396. (21) Dufresne-Martin, G.; Lemay, J.-F.; Lavigne, P.; Klarskov, K. Proteomics 2005, 5, 55-66. (22) Patton, W. F.; Lam, L.; Su, Q.; Lui, M.; Erdjument-Bromage, H.; Tempst, P. Anal. Biochem. 1994, 220, 324-335. (23) Kovarik, A.; Hlubinova, K.; Vrbenska, A.; Prachar, J. Folia Biol. (Praha) 1987, 33, 253-257.

Journal of Proteome Research • Vol. 4, No. 6, 2005 2223

research articles (24) van Oostveen, I.; Ducret, A.; Aebersold, R. Anal. Biochem. 1997, 247, 310-318. (25) Vettermann, C.; Jack, H. M.; Mielenz, D. Anal. Biochem. 2002, 308, 381-387. (26) Kemper, C.; Berggren, K.; Diwu, Z.; Patton, W. F. Electrophoresis 2001, 22, 881-889. (27) Goodman, T.; Schulenberg, B.; Steinberg, T. H.; Patton, W. F. Electrophoresis 2004, 25, 2533-2538. (28) Ge, Y.; Rajkumar, L.; Guzman, R. C.; Nandi, S.; Patton, W. F.; Agnew, B. J. Proteomics 2004, 4, 3464-3467.

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Methogo et al. (29) Steinberg, T. H.; Agnew, B. J.; Gee, K. R.; Leung, W. Y.; Goodman, T.; Schulenberg, B.; Hendrickson, J.; Beechem, J. M.; Haugland, R. P.; Patton, W. F. Proteomics 2003, 3, 11281144. (30) Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999, 294, 13511362. (31) Moore, L. L.; Fulton, A. M.; Harrison, M. L.; Geahlen, R. L. J. Proteome Res 2004, 3, 1184-1190.

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