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GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi.

Nakayasu ES, Yashunsky DV, Nohara LL, Torrecilhas AC, Nikolaev AV, Almeida IC - Mol. Syst. Biol. (2009)

Bottom Line: Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface.TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector.We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968, USA.

ABSTRACT
Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS(n)) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS(n), we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.

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(A) General GPIomic approach. Organic extracts rich in GIPLs and GPI-anchored proteins were obtained as described in Materials and methods. The fraction rich in GIPLs was directly analyzed by LC-MS2 and LC-MS3, whereas the fraction rich in GPI-anchored proteins were digested with proteinase K or trypsin before LC-MS2 and LC-MS3 analyses. In both cases, the LC step was carried out using a POROS R1 10 column (75 μm × 10 cm) and the MS analysis was performed using a LTQXL ESI-linear ion-trap-MS. The resulting spectra were analyzed manually for annotation and assignment of the GPI species. (B) Data-dependent acquisition (DDA) (no dynamic exclusion enabled) LC-MS2 analysis of epimastigote GPIs. The extracted-ion chromatogram was plotted for four major GPI species. The plotted ion species correspond to the dehydrated alkylacylglycerol (AAG—H2O) and the loss of alkylacylglycerol moiety (M—AAG). The insert shows details about the m/z and the area of each peak. (C) Annotated MS2 spectrum from GPI structure at m/z 975.18 corresponding to EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro. (D) MS3 spectrum for the dehydrated AAG fragment at m/z 649.56. (E) Proposed fragmentation and structure of the novel GPI species (EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro) observed at m/z 975.18. Ac, acyl; Gro, glycerol.
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f3: (A) General GPIomic approach. Organic extracts rich in GIPLs and GPI-anchored proteins were obtained as described in Materials and methods. The fraction rich in GIPLs was directly analyzed by LC-MS2 and LC-MS3, whereas the fraction rich in GPI-anchored proteins were digested with proteinase K or trypsin before LC-MS2 and LC-MS3 analyses. In both cases, the LC step was carried out using a POROS R1 10 column (75 μm × 10 cm) and the MS analysis was performed using a LTQXL ESI-linear ion-trap-MS. The resulting spectra were analyzed manually for annotation and assignment of the GPI species. (B) Data-dependent acquisition (DDA) (no dynamic exclusion enabled) LC-MS2 analysis of epimastigote GPIs. The extracted-ion chromatogram was plotted for four major GPI species. The plotted ion species correspond to the dehydrated alkylacylglycerol (AAG—H2O) and the loss of alkylacylglycerol moiety (M—AAG). The insert shows details about the m/z and the area of each peak. (C) Annotated MS2 spectrum from GPI structure at m/z 975.18 corresponding to EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro. (D) MS3 spectrum for the dehydrated AAG fragment at m/z 649.56. (E) Proposed fragmentation and structure of the novel GPI species (EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro) observed at m/z 975.18. Ac, acyl; Gro, glycerol.

Mentions: To ultimately increase the sensitivity and the speed of our analysis, we packed a nanocapillary column with POROS R1 resin and coupled it to an LC-MS system. Then, we performed LC-MS2 and LC-MS3 analysis of organic (91% n-butanol, and combined chloroform/methanol and chloroform/methanol/water) extracts enriched for GIPLs (Figure 3A). GPIs are frequently analyzed in negative-ion mode ESI-MS, dissolved in solutions/buffers with neutral pH. However, under these conditions, the silica tubing has negative charges, which interact with amine (GlcN, AEP, and/or EtNP) groups present in the GPI. To neutralize the negative charge of the silica tubing, we analyzed our GPI samples in positive-ion mode ESI-MS, in the presence of 0.2% formic acid (FA), used as the ion pair for the LC-MS analysis. With the LC-MS approach, we could detect and characterize at least 78 doubly charged ion species of GIPLs (Table II; Supplementary Table I; Supplementary Figure 2A–C). Of those, 70 were found to be novel species (Supplementary Table I). The extracted-ion chromatograms for GIPLs obtained by either extraction procedure (9% n-butanol or combined chloroform/methanol and chloroform/methanol/water) (Figure 3A) clearly indicate that the majority (92–98%) of GIPLs contain ceramide (Cer) and only a small fraction (2–8%) have alkylacylglycerol (AAG) in the lipid moiety (Table II). In MS2, typically the fragmentation (collision-induced dissociation, CID) of the doubly charged GIPL parent-ion gave rise to fragment- or daughter-ions corresponding to the loss of Cer ([M−Cer+H]+), and to a series of ions containing AEP or EtNP residue attached to multiple (n=1–5) hexose (Hex) residues (e.g., Supplementary Figure 2C3, 2C5, 2C7, and 2C9). On the other hand, fragment-ions corresponding to the neutral loss of Hex residue(s) and AEP or EtNP were also highly abundant. We could also find a fragment corresponding to the inositolphosphate (InsP) attached to AEP-HexN (AEP-HexN-InsP) at m/z 529.3 (e.g., Supplementary Figure 2C3, 2C5, 2C7, and 2C9). Interestingly, some species had only one AEP residue and no EtNP. In these species, the AEP residue could be attached to either a Hex or HexN residue. For instance, the fragmentation of the GIPL species AEP-Hex6-HexN-InsP-C16:0/d18:0-Cer observed at m/z 1012.49 showed diagnostic fragment ions corresponding to the AEP-HexN attached to IPC (AEP-HexN-IPC) (m/z 1050.48) and Hex2-AEP (m/z 432.22) (Supplementary Figure 2C22).


GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi.

Nakayasu ES, Yashunsky DV, Nohara LL, Torrecilhas AC, Nikolaev AV, Almeida IC - Mol. Syst. Biol. (2009)

(A) General GPIomic approach. Organic extracts rich in GIPLs and GPI-anchored proteins were obtained as described in Materials and methods. The fraction rich in GIPLs was directly analyzed by LC-MS2 and LC-MS3, whereas the fraction rich in GPI-anchored proteins were digested with proteinase K or trypsin before LC-MS2 and LC-MS3 analyses. In both cases, the LC step was carried out using a POROS R1 10 column (75 μm × 10 cm) and the MS analysis was performed using a LTQXL ESI-linear ion-trap-MS. The resulting spectra were analyzed manually for annotation and assignment of the GPI species. (B) Data-dependent acquisition (DDA) (no dynamic exclusion enabled) LC-MS2 analysis of epimastigote GPIs. The extracted-ion chromatogram was plotted for four major GPI species. The plotted ion species correspond to the dehydrated alkylacylglycerol (AAG—H2O) and the loss of alkylacylglycerol moiety (M—AAG). The insert shows details about the m/z and the area of each peak. (C) Annotated MS2 spectrum from GPI structure at m/z 975.18 corresponding to EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro. (D) MS3 spectrum for the dehydrated AAG fragment at m/z 649.56. (E) Proposed fragmentation and structure of the novel GPI species (EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro) observed at m/z 975.18. Ac, acyl; Gro, glycerol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683718&req=5

f3: (A) General GPIomic approach. Organic extracts rich in GIPLs and GPI-anchored proteins were obtained as described in Materials and methods. The fraction rich in GIPLs was directly analyzed by LC-MS2 and LC-MS3, whereas the fraction rich in GPI-anchored proteins were digested with proteinase K or trypsin before LC-MS2 and LC-MS3 analyses. In both cases, the LC step was carried out using a POROS R1 10 column (75 μm × 10 cm) and the MS analysis was performed using a LTQXL ESI-linear ion-trap-MS. The resulting spectra were analyzed manually for annotation and assignment of the GPI species. (B) Data-dependent acquisition (DDA) (no dynamic exclusion enabled) LC-MS2 analysis of epimastigote GPIs. The extracted-ion chromatogram was plotted for four major GPI species. The plotted ion species correspond to the dehydrated alkylacylglycerol (AAG—H2O) and the loss of alkylacylglycerol moiety (M—AAG). The insert shows details about the m/z and the area of each peak. (C) Annotated MS2 spectrum from GPI structure at m/z 975.18 corresponding to EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro. (D) MS3 spectrum for the dehydrated AAG fragment at m/z 649.56. (E) Proposed fragmentation and structure of the novel GPI species (EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O-acyl-C24:0-Gro) observed at m/z 975.18. Ac, acyl; Gro, glycerol.
Mentions: To ultimately increase the sensitivity and the speed of our analysis, we packed a nanocapillary column with POROS R1 resin and coupled it to an LC-MS system. Then, we performed LC-MS2 and LC-MS3 analysis of organic (91% n-butanol, and combined chloroform/methanol and chloroform/methanol/water) extracts enriched for GIPLs (Figure 3A). GPIs are frequently analyzed in negative-ion mode ESI-MS, dissolved in solutions/buffers with neutral pH. However, under these conditions, the silica tubing has negative charges, which interact with amine (GlcN, AEP, and/or EtNP) groups present in the GPI. To neutralize the negative charge of the silica tubing, we analyzed our GPI samples in positive-ion mode ESI-MS, in the presence of 0.2% formic acid (FA), used as the ion pair for the LC-MS analysis. With the LC-MS approach, we could detect and characterize at least 78 doubly charged ion species of GIPLs (Table II; Supplementary Table I; Supplementary Figure 2A–C). Of those, 70 were found to be novel species (Supplementary Table I). The extracted-ion chromatograms for GIPLs obtained by either extraction procedure (9% n-butanol or combined chloroform/methanol and chloroform/methanol/water) (Figure 3A) clearly indicate that the majority (92–98%) of GIPLs contain ceramide (Cer) and only a small fraction (2–8%) have alkylacylglycerol (AAG) in the lipid moiety (Table II). In MS2, typically the fragmentation (collision-induced dissociation, CID) of the doubly charged GIPL parent-ion gave rise to fragment- or daughter-ions corresponding to the loss of Cer ([M−Cer+H]+), and to a series of ions containing AEP or EtNP residue attached to multiple (n=1–5) hexose (Hex) residues (e.g., Supplementary Figure 2C3, 2C5, 2C7, and 2C9). On the other hand, fragment-ions corresponding to the neutral loss of Hex residue(s) and AEP or EtNP were also highly abundant. We could also find a fragment corresponding to the inositolphosphate (InsP) attached to AEP-HexN (AEP-HexN-InsP) at m/z 529.3 (e.g., Supplementary Figure 2C3, 2C5, 2C7, and 2C9). Interestingly, some species had only one AEP residue and no EtNP. In these species, the AEP residue could be attached to either a Hex or HexN residue. For instance, the fragmentation of the GIPL species AEP-Hex6-HexN-InsP-C16:0/d18:0-Cer observed at m/z 1012.49 showed diagnostic fragment ions corresponding to the AEP-HexN attached to IPC (AEP-HexN-IPC) (m/z 1050.48) and Hex2-AEP (m/z 432.22) (Supplementary Figure 2C22).

Bottom Line: Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface.TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector.We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968, USA.

ABSTRACT
Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS(n)) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS(n), we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.

Show MeSH
Related in: MedlinePlus