Site Specific Phosphorylation of Insulin-Like Growth Factor Binding

Sep 4, 2009 - Insulin-like growth factor binding protein-1 (IGFBP-1) is one of the major insulin-like ... but not pSer169 nor pSer98 of the previously...
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Site Specific Phosphorylation of Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) for Evaluating Clinical Relevancy in Fetal Growth Restriction Majida Abu Shehab,† Shinobu Inoue,‡ Victor K. M. Han,†,§,| and Madhulika B. Gupta*,†,§,| Department of Pediatrics, University of Western Ontario, London, Ontario, Canada, Department of Pediatrics, National Hospital Organization Miyazakihigashi, 4374-1 Tayoshi Miyazaki, Japan 880-0911, and Department of Biochemistry, Children’s Health Research Institute, University of Western Ontario, London, Ontario N6C 2V5 Canada Received March 9, 2009

Fetal growth restriction (FGR) is a leading cause of fetal and neonatal morbidity and mortality. Insulinlike growth factor binding protein-1 (IGFBP-1) is one of the major insulin-like growth factor (IGF) binding proteins involved in fetal growth and development. Our recent data shows that phosphorylation of IGFBP-1 carries both functional and biological relevance in FGR. Considering that IGFBP-1 phosphorylation can be valuable in diagnostics, we examined strategies to enrich IGFBP-1 so that its phosphorylation sites could be assessed by mass spectrometry (MS). Using n ) 3). Additionally, the total IGFBP-1 recovery in the same depleted fractions was further confirmed by IEMA (n ) 3). The quantitation of residual albumin of the samples (n ) 3) was performed by albumin ELISA. 3. Mass Spectrometry for Characterization of IGFBP-1 Phosphorylation. Following 1-D and 2-D SDS-PAGE of the samples (n ) 3), the gel images were acquired as described previously. Guided by an immunoblot, IGFBP-1 band from 1-D gel, or the spots corresponding to IGFBP-1 on 2-D gels stained with SYPRO Ruby were manually excised. The gel slices were cut (1 mm3), immediately, transferred to siliconized Eppendorf tubes, and destained. For in-solution digestion, samples in ammonium bicarbonate (5 mM) buffer were used directly. The peptides were reduced, alkylated and digested with Asp-N (Sigma) (12.5 ng/mL) followed by Trypsin (Promega) (12.5 ng/ mL). The digested peptides were extracted with 5% formic acid and dried in SpeedVac. Samples were either stored at -20 °C or processed directly. Phosphorylated IGFBP-1 peptides were enriched using TiO2. resin. The details for TiO2 based phosphopeptide enrichment and all other MS analysis are described previously.5 In brief, both LC-MS/MS and LC-MS experiments of the enriched samples (n ) 3) were analyzed on a CapLC (Waters, Milford, MA) coupled with a QTOF mass spectrometer (Global Ultima, Micromass, Manchester, U.K.). The phosphopeptide concentrations were determined under identical instrument settings. The selected ion chromatograms for different phosphopeptide peaks were plotted and the spectra summed. The phosphopeptide peak intensities in the summed spectra were assessed for the semiquantitative analysis of the relative levels of the phosphopeptides. LC-MS/MS spectra were processed using the Masslynx software (Version 4.0) and database searches Journal of Proteome Research • Vol. 8, No. 11, 2009 5327

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Figure 1. Detection of IGFBP-1 and its phosphoisoforms in amniotic fluid: Western immuno and ligand blot analyses demonstrating total IGFBP-1 and its phosphoisoform patterns in amniotic fluid. Shown are the immunoblots using anti-IGFBP-1 polyclonal antibody (A, B, and E), while ligand blots using biotin labeled rIGF-I (C and D). Samples in (A and C) are amniotic fluid (total protein, ∼5 µg) (lane 1) and pure IGFBP-1 (total IGFBP-1, 1 ng) (GroPep) (lane 2). The IGFBP-1 phosphoisoform pattern in amniotic fluid (total protein 25 µg) was detected by 2-D immunobloting (B) and 2-D ligand bloting (D). In addition, an aliquot of amniotic fluid (total protein 25 µg) was treated with alkaline phosphatase (ALP) and analyzed by 2-D immunoblot (E).

were conducted using PEAKS software (Bioinformatics Solution, Inc. Waterloo, ON, Canada, version 4.2) or Mascot (http:// mascot.bio.nrc.ca/search_e.php, Matrix Science, Boston, MA) against Swiss-Prot database (version 51.3) for protein identification. Manual verifications confirmed the identified IGFBP-1 phosphopeptides and the respective phosphorylation sites.

Results Detection of IGFBP-1 and Its Isoforms on Immunoblots Using IGFBP-1 Polyclonal Antibody. For assessment of total protein, amniotic fluid sample was analyzed on 1-D SDS-PAGE gel stained with SYPRO Ruby (data not shown). The major protein detected in amniotic fluid was of the higher molecular weight (∼55-65 kDa) corresponding to mainly albumin. IGFBP1, in relatively low abundance, hence, was undetectable by SYPRO Ruby staining. Shown in Figure 1 A is 1-D Western 5328

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Shehab et al. immuno blot using IGFBP-1 polyclonal antibody. IGFBP-1 was detected as an intense band migrating at ∼28 kDa (lane 1) and its proteolytic fragment (∼20 kDa) as described previously.23 Pure IGFBP-1 (GroPep, Thebarton Australia) was used as a reference (shown in lane 2). 2-D immunoblot analysis with the amniotic fluid sample was performed using IGFBP-1 polyclonal antibody to evaluate its isoforms. Several spots (encircled) between 30 and 35 kDa in the pH range of 4.5-5.5 with slightly upward-shifts were visualized in Figure 1B. These spots represent potentially different IGFBP-1 isoforms separated due to phosphorylation of the protein. The ligand blot analysis in Figure 1C with biotin labeled rIGF-I confirmed IGFBP-1 as the major IGF binding protein (∼28 kDa band, lane 1) in amniotic fluid. IGFBP-1 in the commercially purified preparation (lane 2) was not detectable on the ligand blot for reasons unknown to us. 2-D ligand blot analysis of the same sample (Figure 1D) indicated similar spot patterns as with 2-D immunoblot (Figure 1B), but the spots were more diffused. Alkaline phosphatase (ALP) treatment of the amniotic fluid sample eliminated spots on the 2-D immunoblot (Figure 1E). Alignment of spots between ALP treated and the untreated samples confirmed that the removal of spots from the lower pH region was due to dephosphorylation of IGFBP-1. Detection of IGFBP-1 and Its Isoforms on the Immunoblots Using IGFBP-1 Mab 6303 Antibody. Prior to using Mab 6303 to immunoprecipitate IGFBP-1, for comparison, we tested Mab 6303 for its efficiency in detecting IGFBP-1 on 1-D immunoblots. Shown in Figure 2A is amniotic fluid (lane 1) and pure IGFBP-1 (lane 2). The band corresponding to IGFBP-1 (∼28 kDa) was similar to that shown with the polyclonal antibody (Figure 1A). The lower molecular weight (∼20 kDa) fragment was also detectable. We further compared IGFBP-1 isoform patterns separated in amniotic fluid on 2-D immunoblots using Mab 6303. Distribution of multiple spots detected by Mab 6303 was rather scattered at ∼30 kDa (not shown). In addition, the spots were more prominent toward the alkaline region (between pH 5 and 7) rather than the expected for IGFBP-1 (pH 4.5-5.5) as shown earlier with polyclonal antibody in Figure 1B. Additionally, spots were also detected in the lower region (∼25 kDa), but the patterns identified with Mab 6303 were very different from IGFBP-1 polyclonal (Figure 1B). The discrepancy in these patterns suggested that, although Mab 6303 was highly specific when used in conjunction with 1-D immunoblotting, it reacted nonspecifically on 2-D immunoblots. We presumed this to be due to the sample preparation strategy used for 2-D gel based separations. A. IGFBP-1 Enrichment Using Antibody Based Techniques. 1. Immunoprecipitation. The use of Mab 6303 in detecting IGFBP-1 with immunobloting and also its use in immunoprecipitation has been well-documented.6-8 Considering the high specificity and sensitivity of Mab 6303 with 1-D immunoblot analysis recorded in detecting IGFBP-1 also in our study (Figure 2A), we used Mab 6303 for immunoprecipitation of IGFBP-1. Mab 6303 was also used as the primary antibody to detect IGFBP-1 on 1-D immunoblots. Following immunoprecipitation, IGFBP-1 eluted using different buffers (as described in Material and Methods) is shown in Figure 2B. Similar to amniotic fluid (used as a positive control, lane 2), the immunoprecipitated samples showed IGFBP-1 at ∼28 kDa (lanes 3-5), as determined by the molecular size marker in lane 1. The band corresponding to IGFBP-1 in the samples eluted with SDS

IGFBP-1 Phosphorylation Characterized Proteomically

Figure 2. Enrichment of IGFBP-1 by immunoprecipitation: Western immuno blot analyses demonstrating total IGFBP-1 in amniotic fluid using anti IGFBP-1 monoclonal antibody (Mab 6303). Shown in (A) is 1-D immunoblot of amniotic fluid (total protein ∼5 µg) (lane 1) and pure IGFBP-1 (GroPep) (1 ng of IGFBP-1) (lane 2). IGFBP-1 Mab 6303 was used to immunoprecipitate IGFBP-1 from amniotic fluid. The immunoblot in (B) was performed using Mab 6303 with amniotic fluid (5 µg of total protein, lane 2). Equal aliquot of the immunoprecipitated IGFBP-1 eluted with (i) SDS loading buffer, lane 3; (ii) Urea, CHAPS buffer with DTT, lane 4; and (iii) Urea and CHAPS buffer without DTT, lane 5; the molecular weight marker is illustrated in lane 1. Additionally, for comparison, the previous immunoblot performed using Mab 6303, shown in (B), was stripped and reanalyzed using antihuman IGFBP-1 polyclonal antibody (C). Samples are amniotic fluid (total protein ∼5 µg), Lane 2 and immunoprecipitated IGFBP-1 eluted with (i) SDS buffer, lane 3; (ii) Urea, CHAPS and DTT buffer, lane 4; and (iii) Urea, CHAPS buffer without DTT, lane 5. An aliquot of the immunoprecipitated IGFBP-1 eluted with Urea, CHAPS and DTT (C, lane 4) was subjected to 2-D immunoblot analysis using anti-IGFBP-1 polyclonal antibody is shown in (D). For detection of IgG coeluted with IGFBP-1 during immunoprecipitation, the immunoblot in (B) was stripped and reanalyzed using anti-human IgG polyclonal antibody. The IgG heavy (H) and light (L) chain proteins present in the immunoprecipitate are shown in (E).

buffer (lane 3) and with urea and CHAPS buffer with DTT (lane 4) was faint. The intensity for IGFBP-1 eluted using urea and CHAPS buffer without DTT (lane 5) was nonetheless highest. In the same sample (lane 5), we also detected an additional band at ∼90 kDa. This band was attributed to multimeric form

research articles of IGFBP-1 as has been reported previously.24 Since this multimer was eliminated by the use of DTT (lane 4), further analysis of the corresponding protein was considered not as significant. Identification of IGFBP-1 in the immunoprecipitated sample (eluate, urea CHAPS without DTT) by 2-D immunoblot using Mab 6303 antibody showed no defined spots as were observed earlier with the direct amniotic fluid (data not shown). This analysis confirmed that Mab 6303 reacted nonspecifically with other proteins in the samples resulting in aberrant spot patterns on 2-D immunoblots. Detection of IGFBP-1 in the Immunoprecipitate Using IGFBP-1 Polyclonal Antibody. Our next approach was thus to evaluate whether IGFBP-1 in the immunoprecipitate was detectable by IGFBP-1 polyclonal antibody. 1-D immunoblot (shown in Figure 2B) performed using Mab 6303 (as the primary antibody) was stripped and re-evaluated with IGFBP-1 polyclonal antibody. As explained above and as compared to amniotic fluid (lane 2) and the molecular weight marker (lane 1), the faint band corresponding to IGFBP-1 eluted using SDS buffer remained the same, shown in Figure 2C, lane 3. The sample eluted using urea and CHAPS buffer with DTT (Figure 2C, lane 4), nonetheless, showed a significant difference in the intensity as well the mobility of IGFBP-1 band as compared to observed with the Mab 6303 antibody (Figure 2B, lane 4). In the presence of DTT (Figure 2C, lane 4), IGFBP-1 polyclonal antibody detected a band corresponding to IGFBP-1 that was slightly higher in molecular size and in intensity. The differences observed with the size of the band on the immunoblot suggest that IGFBP-1 in amniotic fluid migrates at ∼28 kDa on SDS-PAGE under nonreducing conditions (Figure 1A), while at ∼34 kDa under reducing conditions (with DTT) (Figure 2C, lane 4). Similar findings have been reported previously for IGFBP-1 under these conditions.25 Additionally, to compare IGFBP-1 isoform patterns in the immunoprecipitated IGFBP-1, sample with DTT (shown in Figure 2C, lane 4) was subsequently analyzed by 2-D immunobloting using IGFBP-1 polyclonal antibody. The 2-D spot pattern for IGFBP-1 (Figure 2D) on the immunoblot using IGFBP-1 polyclonal antibody matched with the immunoblot performed using direct amniotic fluid (depicted in Figure 1B). The spots in the encircled area were within the expected pH range (4.5-5.5) showing an upward shift toward the acidic pH confirmed IGFBP-1 phosphorylation. These data led us to conclude that Mab 6303 was affected by the reducing conditions used in 2-D SDS-PAGE sample preparation procedure. We henceforth restricted the use of Mab 6303 in detection of IGFBP-1 on 1-D immunoblots, in addition to immunoprecipitation. Because of interference with the buffers used to elute IGFBP-1 from the immunoprecipitate, quantitation of IGFBP-1 by IEMA was not performed. The recovery of IGFBP-1 in the samples from 1-D immunoblots (Figure 2C, lanes 3-5) was therefore evaluated semiquantitatively by densitometry. After careful comparison with the amniotic fluid used as a reference, IGFBP-1 recovery in samples shown in lanes 4 and 5 was estimated as ∼30% (data not shown). Considering classical immunoprecipitation could accompany IgG heavy (H) and light (L) chain contaminants, we determined whether in our samples elution of IGFBP-1 from the immunoprecipitate co-eluted and enriched IgGs. We stripped the PVDF membrane (used previously, shown in Figure 2B) and re-evaluated it with anti-IgG antibody. As expected, shown in Journal of Proteome Research • Vol. 8, No. 11, 2009 5329

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Figure 3. Seize immunoaffinity purification of IGFBP-1: Immunoblot analysis of Seize immunoaffinity purified IGFBP-1 using Mab 6303. (A) Amniotic fluid (total protein ∼5 µg), lane 2 and Seize immunoaffinity purified IGFBP-1 (5 µL) fractions nos. 1 and 2, lanes 3 and 4. Both intact IGFBP-1 (∼28 kDa band) and the fragmented IGFBP-1 (∼20 kDa band) were visualized. A representative ligand blot using biotin labeled rIGF-I with Seize immunoaffinity purified IGFBP-1 is shown in panel B. Amniotic fluid (5 µg of total protein) is shown in lane 2, and Seize immunoaffinity IGFBP-1 fractions nos. 1 and 2 (5 µL each) are shown in lane 3 and 4, respectively. 2-D immunoblot using anti-IGFBP-1 polyclonal antibody with purified IGFBP-1 (∼50 uL) performed using fraction no. 2 is illustrated in panel C. Aliquots of Seize immunoaffinity purified IGFBP-1 were further electrophoresed on 1-D SDS-PAGE (∼100 ng) (D), and on 2-D SDS-PAGE (∼200 ng) (E) stained with SYPRO Ruby. The band corresponding to IGFBP-1 from 1-D gel (D) and spots nos. 1-7 from 2-D SDS gel (E) were excised for in-gel digestion and IGFBP-1 phosphopeptide mapping performed. MS data with samples from 1-D gel are shown in panel F and with 2-D gel in panel G that are representative of relative phosphopeptide intensities for the identified IGFBP-1 phosphorylation sites from the two respective sets of Seize immunoaffinity samples.

Figure 2E (lanes 4 and 5) are multiple bands corresponding to IgG (H) and (L) chain isotypes with variable intensities. These results confirmed coelution of high amounts of IgGs with the immunoprecipitate and their interference with immunobloting for IGFBP-1. Identification of IGFBP-1 Phosphopeptides in Immunoprecipitated Sample by LC-MS/MS. Following immunoprecipitation, the samples eluted with urea CHAPS with DTT buffer were selected for MS identification of IGFBP-1 phosphorylation sites. Samples for MS analysis were analyzed using both in-gel and in-solution techniques. Several attempts to identify IGFBP-1 phosphopeptides with the immunoprecipitated samples were not successful. As expected, the high concentrations of H and L chains of IgG isotypes coeluted with the immunoprecipitated IGFBP-1 resulted in masking effects. MS analysis thusly failed to identify IGFBP-1 in the immunoprecipitated samples. 2. IGFBP-1 Enrichment Using Seize Immunoprecipitation Technology. Detection of IGFBP-1 and Its Isoforms on Immunoblots. Seize immunoaffinity resin benefited in our ability to purify/enrich IGFBP-1 without coenriching IgGs. The covalently coupled Mab 6303 with the resin retained both H and L chain IgG isotypes. Detection of IGFBP-1 in Seize affinity IGFBP-1 sample on 1-D immunoblot was performed and showed that 5330

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the smaller IGFBP-1 fragment (∼20 kDa band) (Figure 3A, lane 3) preceded the intact IGFBP-1. Shown in Figure 3A, lane 4 is the pooled subsequent fractions following the proteolyzed fragment that constituted mainly the intact IGFBP-1 (∼28 kDa). Semiquantitative densitometric analysis of several 1-D immunoblots (Figure 3A, lane 4) provided ∼70% recovery of IGFBP-1 from at least two different Seize immunoaffinity batches (data not shown). The Seize affinity purified IGFBP-1 was also detectable on the ligand blot (rIGF-I) (Figure 3B) suggesting that the protein retained its IGF-I binding capacity. Since IGF-I binding requires intact protein, as reported previously,23 the fragmented IGFBP-1 was not detectable on ligand bloting. Subsequently, IGFBP-1 isoforms in the Seize affinity purified IGFBP-1 sample (Fraction no. 2) (Figure 3A, lane 4) were evaluated by 2-D immunoblot analysis. Multiple spots were detected that corresponded with the intact IGFBP-1 as well the fragmented IGFBP-1 peptide, but were in tightly clustered form (Figure 3C). LC-MS/MS Identification of IGFBP-1 Phosphopeptides in Seize Immuno Affinity Isolated Samples. MS identification of IGFBP-1 phosphorylation sites was performed using the Mab 6303 based-Seize affinity purified IGFBP-1 (Fraction 2) as

IGFBP-1 Phosphorylation Characterized Proteomically shown earlier in Figure 3A, lane 4. In-gel digestion was performed with the intact IGFBP-1 excised from 1-D gel stained with SYPRO Ruby depicted in Figure 3D (lanes 2 and 3) and 2-D gel (Figure 3E). After dual digestion and TiO2 enrichment, two phosphorylation sites, pSer101 and pSer119 (Figure 3F) were detected by LC-MS/MS analysis. In-solution digests of the sample (n ) 3) revealed similar results. Multiple spots were detected with the Seize affinity purified IGFBP-1 on 2-D immunoblots (Figure 3E). We thus also performed MS analysis using samples extracted from individual spots (nos.1-7) on SYPRO Ruby stained 2-D gels (shown in Figure 3E). Our LCMS/MS data from spot nos. 1-7 (Figure 3G) represents potentially different IGFBP-1 isoforms separated from basic to acidic pH (pH 5.5-4.5) on the 2-D gel. Two major IGFBP-1 phosphorylation sites, pSer101 and pSer119, were detected that corresponded to spot numbers 2, 3 and 4, while no IGFBP-1 phosphopeptide was detected from spot 1. The absence of IGFBP-1 phosphopeptides corresponding to the most basic spot (no. 1) suggests that this spot may represent non/or the least phosphorylated form of IGFBP-1, or alternatively with levels of phosphorylation below the threshold of detection. While the intensity for spot (5) (the proteolytic fragment) was very high on the gel, none of the phosphorylation sites were detectable in this fraction. We presume that the migration of this spot (5) toward the most acidic pH may be due to the acidic characteristic of the proteolytic peptide and not because of the higher level of phosphorylation. We further evaluated the Seize immunoaffinity sample to determine the relative IGFBP-1 phosphopeptide intensities for the two sites. The LC-MS data demonstrated both pSer101 and pSer119 sites with high peak intensities. On the basis of our MS data, we succeeded in detecting the two major IGFBP-1 phosphorylation sites, pSer101 and pSer119, using the Seize affinity purified samples. However, our objective in this study was to identify all known (four) phosphorylation sites. To overcome this constraint, we sought alternate procedures for enrichment of IGFBP-1. B. Non-Antibody Based Strategy for Enrichment of IGFBP-1. 1. IGFBP-1 Separation in ZOOM Fractionated Samples under Denaturing Conditions. The small-scale device ZOOM IEF Fractionator was a substitute to relying on antibodies to enrich IGFBP-1. ZOOM technology was most suitable because it uses a gel-free fractionation system. Following ZOOM IEF, total protein contents in fractions F1-F5 were analyzed on 1-D SDSPAGE and SYPRO Ruby staining. In Figure 4A, the intensity of the bands using equal aliquots of samples shows that more than ∼50% of the higher molecular weight proteins, such as albumin, were visualized in fraction no. 3 (F3). Upon the basis of the size of the band, we concluded that F3 (pH 5.3-6.2) contained mainly albumin, the pI for which corresponds with the predicted pI of 5.92. Immunoblot analysis of the fractions using Mab 6303 antibody indicated that IGFBP-1 was enriched mainly into two fractions, F1 and F2 (Figure 4B). The bulk of IGFBP-1 was in fraction F2 (pH 4.6-5.3) close to the pI for IGFBP-1 (equal to around 5.0). The densitometric analysis of the immunoblot showed ∼30% recovery of IGFBP-1 in these two fractions (data not shown). Furthermore, in order to determine whether proteins fractionated by ZOOM IEF could be separated by 2-D SDS-PAGE, an aliquot of fraction F2 (with majority of IGFBP1, pH 4.6-5.3) was analyzed using 2-D SDS-PAGE. As illustrated in Figure 4C, while most of albumin was separated in fraction F3 and the bulk of IGFBP-1 in F2, an overlap due to high

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Figure 4. Enrichment of IGFBP-1 by solution phase isoelectric focusing (IEF): Amniotic fluid sample (∼2 mg of total protein) was fractionated using ZOOM IEF. Equal volume (30 µL) of ZOOM fractionated samples (ZOOM fractions) were electrophorsed on 1-D SDS-PAGE and stained with SYPRO Ruby (A). Shown is also 1-D immunoblot using IGFBP-1 Mab 6303 (B) with amniotic fluid (AF) (5 µg of total protein, 1 ng of IGFBP-1), lane 1, and unfractionated (Pre-ZOOM) amniotic fluid sample prepared for IEF in lane 2. Additionally are shown five different fractions (F1-F5) collected following ZOOM fractionation. F1 with pH 3-4.6, F2 with pH 4.6-5.3, F3 with pH 5.3-6.2, F4 with pH 6.2-7, and F5 with pH 7-10. An aliquot (30 µL) of F2, pH 4.6-5.3 (shown in B) separated with bulk of IGFBP-1 was further analyzed by 2-D SDS-PAGE and stained with SYPRO Ruby (C) and on 2-D immunoblot using anti IGFBP-1 polyclonal antibody (D).

amount of albumin was apparent. SYPRO Ruby stained 2-D gel image confirmed that ZOOM IEF was not successful in generating a good proteome due to cofractionation of albumin. Although several IGFBP-1 isoforms were separated with 2-D immunobloting (Figure 4D), the spots were fewer in number. Because of abundant albumin, the complexity of the fractionation buffers and significantly low recovery of IGFBP-1, MS analysis was not successful. The ineffectiveness of ZOOM IEF technology was evident due to residual albumin in the fraction with IGFBP-1. These findings necessitated removal of albumin as an optimal step for MS analysis. 2. Enrichment of IGFBP-1 by Albumin Depletion. Removal of albumin in amniotic fluid was performed using Affi-Gel Blue. Journal of Proteome Research • Vol. 8, No. 11, 2009 5331

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Figure 5. Enrichment of IGFBP-1 by albumin depletion using AffiGel Blue gel: Following albumin depletion (n ) 3) of an amniotic fluid sample, the depleted and the bound (eluted) fractions were reconstituted to the original sample volume (1 mL) of amniotic fluid used for depletion (Table 1). Equal aliquots (5 µL) of the amniotic fluid (lane 2), the depleted (lane 3) and the bound (eluted) fractions (lane 4) were electrophoresed on 1-D SDS-PAGE and stained with SYPRO Ruby (A). The amniotic fluid (total protein, 25 µg) and albumin depleted sample were also evaluated on 2-D SDS-PAGE stained with SYPRO Ruby (B) and (C), respectively. Further shown is 1-D immunoblot using Mab 6303 (D) with amniotic fluid, lane 2; the depleted sample, lane 3; and the bound (eluted) fraction, lane 4 (total protein, 5 µg each). Densitometry evaluation of total IGFBP-1 recovery from 1-D immunoblots performed with three different albumin depletions (n ) 3) of the same amniotic fluid sample was estimated (E). Representative 2-D immunoblot using anti-IGFBP-1 polyclonal antibody of the depleted sample (F) and the bound (eluted) fraction (G) (total protein, 25 µg each). The relative phosphopeptide peak intensities of LC/MS assessments for one of the three depletion experiments is represented (H).

Figure 5A shows total protein and albumin contents in amniotic fluid before and after depletion. Reduced intensity of the ∼50 kDa band was observed in Figure 5A (lane 3). Results from SYPRO Ruby stained 2-D gels of the samples before and after depletion (Figure 5B,C) depicted an improved separation of amniotic fluid proteome. IGFBP-1 in the depleted samples was evaluated by 1-D immunoblot analysis (Figure 5D). The band corresponding to IGFBP-1 is shown in amniotic fluid (lane 2), the depleted fraction (lane 3) and the bound (eluted) fraction (lane 4). ELISA showed reduction in albumin contents 3.73 × 105 ( 2.35 × 105 ng/mL (n ) 3) which confirmed ∼8.5% residual albumin in the depleted amniotic fluid (Table 1) and (Figure 5A). IGFBP-1 in the depleted sample quantified by IEMA showed a total of 1.36 × 103 ( 0.174 × 103 µg/L which corresponded to ∼68% (n ) 3) recovery (Table 1) of IGFBP-1 that matched densitometric evaluations (Figure 5E). Overall, albumin depletion enhanced the loading capacity on the IPG strips for IEF and provided better separation of 5332

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IGFBP-1 isoforms on 2-D gel. 2-D immunoblot analysis for IGFBP-1 of the depleted sample is shown with multiple spots (Figure 5F). Similar number of spots as with the direct amniotic fluid (shown earlier in Figure 1B) were detected following depletion. Dephosphorylation by ALP treatment confirmed the phosphorylation status of the protein (data not shown). The IEMA evaluation of the samples demonstrated that 2.07 × 102 ( 0.84 × 102 µg/L (∼30%) of IGFBP-1 was likely to be bound to albumin (Table 1). In an attempt to determine whether there was a specific loss of phosphorylated forms, IGFBP-1 bound to albumin (eluted from the bound fraction) was examined by 2-D immunobloting. Only one spot (Figure 5G) on the immunoblot was detected in this sample. These results established that possibly only a small amount of IGFBP-1 remained bound to albumin. Over all, albumin depletion was the most suitable method to enrich IGFBP-1 for conducting downstream MS analysis. Identification of IGFBP-1 Phosphopeptides in Albumin Depleted Sample by LC-MS/MS. Subsequent to dual enzyme

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IGFBP-1 Phosphorylation Characterized Proteomically

Table 1. Quantitative Evaluation of Total Protein, Albumin and IGFBP-1 Following Albumin Depletion of Amniotic Fluid Using Affi-Gel Blue Gela sample (amniotic fluid)

volume (mL)

total protein (mg/mL)

total albumin (ng/mL)

total IGFBP-1 recovery (µg/L)

Direct (crude) Recovery Albumin depletedb Recovery Bound fractionb Recovery

1

2.79 ( 0.29 (100%) 1.71 ( 0.22 (61%) 1.08 ( 0.16 (40%)

4.39 × 106 ( 2.36 × 106 (100%) 3.73 × 105 ( 2.35 × 105 (8.5%) 1.58 × 105 ( 4.85 × 104 (3.5%)

1.97 × 103 ( 0.129 × 102 (100%) 1.36 × 103 ( 0.174 × 103 (68%) 2.07 × 102 ( 0.84 × 102 (10%)

1 1

a The amniotic fluid before and after depletion was evaluated for total protein by BCA. Albumin and IGFBP-1 concentrations were estimated by ELISA and IEMA, respectively. The values are represented as the mean ( SD of different depletions (n)3) of amniotic fluid sample used in this study. The recovery (in parentheses) is represented as percentage of total concentrations compared to amniotic fluid (nondepleted). b For comparison, volumes reconstituted to direct amniotic fluid aliquot (1 mL) used for depletion using Affi gel.

Table 2. The IGFBP-1 Phosphopeptides with Identified Phosphorylation Sites in Albumin Depleted Amniotic Fluid Detected by LC-MS/MS Analysisa phosphorylated residue

m/z

phosphopeptide sequence

Ser101 Ser101/Ser98 Ser119 Ser169

(949.747400, 3+) (979.73) (693.250600, 2+) 629.73

DASAPHAAEAGSPESPEpSTEITEEELL DASAPHAAEAGSPEpSPEpSTEITEEEL DNFHLMAPpSEE A QETpSGEEISK

a Amino acid sequences of the phosphopeptides with four IGFBP-1 serine phosphorylation sites (indicated in bold as pS) identified by LC-MS/MS analysis in amniotic fluid (n)3). The deconvoluted spectra the novel site pSer 98 has been shown earlier with pSer101 singly and with pSer98.

digestion and TiO2 enrichment as described earlier, LC-MS/ MS analysis with both in-solution and in-gel digested samples of albumin depleted amniotic fluid (n ) 3) detected the four expected IGFBP-1 phosphorylation sites. Of these, pSer101, pSer119 and pSer169 were reported in the past17 using recombinant protein expressed in CHO cells. The doubly phosphorylated peptide with pSer98 together with pSer101 has been identified recently in the secretion from HepG2 cells5 and in FFE fractionated amniotic fluid.9 MS data from three separate analysis confirmed identification of four IGFBP-1 phosphorylation sites. Table 2 shows the amino acid sequences of the phosphopeptides and their m/z ratios together with the respective sites. Furthermore, we carried out LC-MS analyses to detect semiquantitatively peak intensities for the phosphopeptides with four identified sites. Figure 5H is a representation (n ) 3) of the relative intensities of four IGFBP-1 phosphopeptides comprising pSer98, pSer101, pSer119 and pSer169 sites, respectively, in one of the estimations. Considering the MS assessments for the phosphorylation intensity were performed at separate times, the data for each of the identified sites is represented as the percentage of phosphopeptide intensity recorded for the pSer101 site (Table 3). The phosphopeptide intensity data following enrichment of the sample by depletion showed pSer119 site and pSer101 to have higher intensities,

while for pSer169 and pSer98 were up to ∼20-fold and ∼10fold lower, respectively, compared to pSer101 (Table 3). Relative assessments of intensities by LC/MS with depleted amniotic fluid corroborate with our previous FFE fractionated amniotic fluid isoforms.9 We thus conclude that to perform differential phosphopeptide mapping for IGFBP-1 phosphorylation using clinical samples, this approach would be the most appropriate in further evaluations to determine its diagnostic applications.

Discussion IGF axis plays an important role in fetal growth and development.26 Phosphorylation of IGFBP-1 has widely been speculated to contribute to perturbation of IGF axis and play a key role in FGR. We recently supported this hypothesis by demonstrating biological relevance of increased site-specific IGFBP-1 phosphorylation in conditions mimicking FGR.5 IGFBP-1 phosphorylation is considered important also in diagnosis of FGR.6,8 Previous studies used immunoassays to link increase in IGFBP-1 phosphorylation with FGR;27-29 however, the data is inconclusive. In the current study, we developed a simple albumin depletion strategy and focused on identifying the four known IGFBP-1 phosphorylation sites in amniotic fluid by MS. The data from this study additionally provided feasibility for the measurement of changes in site-specific IGFBP-1 phosphorylation in FGR. Use of this approach with clinical samples should provide a more accurate reflection of this PTM in FGR and facilitate evaluation of its implications in diagnostics. Native gel electrophoresis has been frequently used to determine IGFBP-1 phosphorylation in biological fluids. The other common approach to examine PTMs for IGFBPs30 has been 2-D ligand blot analysis. To detect phosphoisoforms, specifically for IGFBP-1, we incorporated 2-D immunobloting using IGFBP-1 polyclonal antibody as a basic technique.5 While Mab 6303 was highly effective in immunoprecipitation and most sensitive antibody for detection of IGFBP-1 by 1-D immunoblotting, the Mab 6303 did not show adequate reactivity under reducing conditions necessary for 2-D-PAGE analysis with amniotic fluid in this study.

Table 3. Phosphopeptide Peak Intensity Determined in Albumin Depleted Amniotic Fluid by LC-MS Analysisa

Mean percentage of phosphopeptide peak intensity relative to pSer101 (n ) 3) Fold reduction in phosphopeptide peak intensity relative to pSer101

pSer101

pSer98/pSer101

pSer119

pSer169

100 ( 0%

13.32 ( 3.31%

111.8 ( 27.32%

4.51 ( 6.17%

1

7.5

0.89

22.2

a Mean ((SD) percentage and average fold-change of the phosphopeptide peak intensity for each phosphorylation site determined relative to the intensity of pSer101 (LC-MS) in albumin depleted amniotic fluid sample.

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research articles Amniotic fluid is a dynamic fluid reflective of fetal and maternal blood and the uterine decidua that also contains high levels of IGFBP-1.26 Typically, the concentration of IGFBP-1 in amniotic fluid (2-100 µg/mL)31 is sufficient for analysis of IGFBP-1 by MS; however, only a smaller proportion of IGFBP-1 is phosphorylated. Furthermore, overwhelming amounts of nonphosphorylated proteins including abundant proteins (e.g., albumin and IgG) mask the identification of the low-abundance phosphopeptides by MS and pose a variety of technical challenges.32 Application of in-gel based methodologies for phosphopeptide mapping by MS could be valuable for biofluids.33 The high-abundance proteins in biofluids nonetheless restrict the loading capacity on the gels thus limit identification of low-abundance phosphopeptides.34 Enrichment or purification of total IGFBP-1 therefore was a prerequisite for MS analysis. Immunoprecipitation is the most widely employed technique for rapid enrichment of a protein of interest. Although several anti-IGFBP-1 antibodies have been used to immunoprecipitate IGFBP-1 from biological samples, in selecting a commercially available antibody, we considered Mab 6303, known to capture most of IGFBP-1 phosphoisoforms with high specificity.27 Using Mab 6303 for immunoprecipitation, we nevertheless recognized that it accompanied with high amounts of IgGs in the immunoprecipitate that significantly interfered with MS analysis. It was thus crucial to integrate an immunoaffinity technology that uses direct coupling of the Mab 6303 to the resin. Covalent coupling of Mab 6303 with Seize affinity resin introduced in this study indeed facilitated removal of IgGs from the purified IGFBP-1. Coverage of the expected IGFBP-1 phosphorylation sites with IGFBP-1 affinity purified samples was, nonetheless, not adequate. In spite of repeated MS attempts, we were unable to detect all four expected IGFBP-1 phosphorylation sites. To eliminate ambiguity, we considered fractionation of amniotic fluid using ZOOM IEF as a valuable alternative approach to circumvent antibody based enrichment of IGFBP1. The major disadvantage, on the other hand, remained abundant albumin. Having a similar isoelectric point (pI), albumin was partially cofractionated with IGFBP-1. In addition, the limited loading capacity and the complexity of the sample buffer, together with protein loss during sample preparation technique, cumulatively resulted in lower yield of IGFBP-1. Overall, ZOOM IEF remained inefficient and ineffective with MS analysis for IGFBP-1. We have recently optimized albumin depletion strategy in maternal plasma proteome profiling in FGR pregnancies.35 Depletion strategy has been allocated to study also phosphorylation and other PTMs on proteins.36 Although 25-30% loss of IGFBP-1 was inevitable, based on our repeated assessments (Table 1), the depletion procedure proved to be reproducible in detecting the expected IGFBP-1 phosphorylation sites in amniotic fluid (Tables 2 and 3). Depletion improved 2-D gel resolution of amniotic fluid proteome and subsequently enhanced IGFBP-1 isoform separation. Most importantly, the depleted sample provided high success in identifying the expected IGFBP-1 phosphorylation sites that were not detectable by other procedures tested in this study. Three IGFBP-1 phosphorylation sites, Ser101, Ser119 and Ser169,17 have been reported previously. Additionally, a novel doubly phosphorylated IGFBP-1 peptide with pSer98 and pSer101 was detectable in secretion from HepG2 cells5 and in amniotic fluid, but only following FFE separated isoforms.9 LCMS/MS analysis provided success in identifying these four 5334

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Shehab et al. expected IGFBP-1 phosphorylation sites using a simpler enrichment technique with albumin depletion. In addition to the use of depleted amniotic fluid, it was possible to estimate basic changes in relative peak intensities by LC-MS for each four phosphopeptides. The phosphopeptide peak intensities for the two sites (pSer98 and pSer169) were much lower in the depleted samples relative to pSer101 and pSer119. These findings were substantiated by our previous FFE fractionated amniotic fluid IGFBP-1 phosphoisoforms.9 The significance of relative evaluation of IGFBP-1 phosphorylation status between control and healthy pregnancies is well recognized through our previous report.9 With expectations of increased phosphorylation in FGR pregnancies,9 it is anticipated that the use of IGFBP-1 enriched by albumin depletion should provide feasibility in establishing the much needed link of this PTM with FGR. In summary, this study is the first that discusses the pros and cons of different enrichment methods and their significance to study IGFBP-1 phosphorylation proteomically. In comparing both the antibody based and alternative approaches, we conclude that albumin depletion is most suitable and consistent approach to enrich IGFBP-1. Use of this approach will be vital in establishing a link of IGFBP-1 phosphorylation with FGR and basis for developing phosphorylation site-specific antibodies for diagnosis and insights into its role in FGR pregnancies.

Acknowledgment. We gratefully acknowledge Dr. Gillis Lajoie, Director, Biological Mass Spectrometry Laboratory (BMSL), University of Western Ontario, London, Ontario, Canada, for his keen interest, insight and invaluable discussions during this study. We thank Dr. Suya Liu (BMSL) for his technical and intellectual contributions in conducting MS analysis. MS analysis for this study in part was also conducted by Protana, Inc., Toronto, Ontario, Canada. We are also grateful to Ms. Sylvia Katzer for her valuable time and interest in diligently proofreading and editing this manuscript. M.B.G. received a Natural Sciences and Engineering Research Council of Canada (NSERC)-Discovery Grant for financial support that includes salary support for M.A.S. References (1) Ergaz, Z.; Avgil, M.; Ornoy, A. Reprod. Toxicol. 2005, 3, 301–322. (2) Tisi, D. K.; Liu, X. J.; Wykes, L. J.; Skinner, C. D.; Koski, K. G. J. Nutr. 2005, 7, 1667–1672. (3) Ong, K.; Kratzsch, J.; Kiess, W.; Costello, M.; Scott, C.; Dunger, D. J. Clin. Endocrinol. Metab. 2000, 11, 4266–4269. (4) Sheridan, C. Aust. Fam. Physician 2005, 9, 717–723. (5) Seferovic, M. D.; Ali, R.; Kamei, H.; Liu, S.; Khosravi, J. M.; Nazarian, S.; Han, V. K.; Duan, C.; Gupta, M. B. Endocrinology 2009, 1, 220– 231. (6) Westwood, M.; Gibson, J. M.; Davies, A. J.; Young, R. J.; White, A. J. Clin. Endocrinol. Metab. 1994, 6, 1735–1741. (7) Westwood, M.; Gibson, J. M.; White, A. Endocrinology 1997, 3, 1130–1136. (8) Martina, N. A.; Kim, E.; Chitkara, U.; Wathen, N. C.; Chard, T.; Giudice, L. C. J. Clin. Endocrinol. Metab. 1997, 6, 1894–1898. (9) Nissum, M.; Abu Shehab, M.; Sukop, U.; Khosravi, J. M.; Wildgruber, R.; Eckerskorn, C.; Han, V. K.; Gupta, M. B. Mol. Cell. Proteomics 2009, 8 (6), 1424–1435. (10) Barker, D. J. J. Hypertens. Suppl. 1992, 7, S39–44. (11) Barker, D. J. Horm. Res. 2005, 2–7. (12) Barker, D. J.; Martyn, C. N. J. Epidemiol. Community Health 1992, 1, 8–11. (13) Gibson, J. M.; Westwood, M.; Lauszus, F. F.; Klebe, J. G.; Flyvbjerg, A.; White, A. Diabetes 1999, 2, 321–326. (14) Miell, J. P.; Jauniaux, E.; Langford, K. S.; Westwood, M.; White, A.; Jones, J. S. Mol. Hum. Reprod. 1997, 4, 343–349. (15) Fowler, D.; Albaiges, G.; Lees, C.; Jones, J.; Nicolaides, K.; Miell, J. Hum. Reprod. 1999, 11, 2881–2885.

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