3.4. Analysis of the Phosphorylation Status of the 11–24 Proteins11–24 Proteins
3.4.3. Phosphorylation Sites Identification
Röhriget al.(2006) reported the phosphorylation sites of CDeT11–24. They used a Metal Oxide Affinity Chromatography (MOAC) approach to enrich phosphoproteins fromC. plan-tagineum followed by 2D–PAGE and LC–MS/MS analysis to identify the phosphorylation sites of CDeT11–24.
Due to the sub-stoichiometry of phosphorylated proteins in complex mixtures and the suppression of non-phosphorylated peptides over phosphorylated peptides during MS anal-ysis, often an enrichment step is required for the identification of phosphorylation sites.
Sugiyamaet al.(2007) reported an improvement of the phosphopeptide enrichment based
on TiO2 Metal Oxide Chromatography (MOC) with aliphatic hydroxy acids as competitors for improving the specificity of the phosphopeptide isolation.
Here this approach was used for isolating the phosphopeptides of the CDeT11–24 protein to compare them with the ones reported by Röhrig et al. (2006). Additionally, the MOC enrichment was performed on the Lb11–24 and Ls11–24 homologue to confirm the results from the immunoprecipitation experiments (section3.4.1) and the 2D shift assays (section 3.4.2).
Craterostigma plantagineum CDeT11–24 Phosphopeptide Enrichment and Phosphorylation Sites Identification
CDeT11–24 from C. plantagineum dried leaves was immunochromatographically purified using an IgG-coupled affinity column. Affinity-purified CDeT11–24 was digested with trypsin and subjected to phosphopeptide enrichment on TiO2 columns. Nano–LC–MS/MS analysis of phosphopeptide-enriched tryptic digests yielded three phosphopeptides (Figure 3.20) with mass-to-charge ratios (m/z) of 686.36 (2+), 414.72 (2+) and 555.31 (2+), corresponding to the phosphorylated forms of tryptic peptides T15, T30 and T36 (Table 3.5). Alternative and equivocal phosphorylation sites are marked by letters.
Fragmentation spectra are illustrated in Figure 3.21 for peptide T15 and its three differ-ent alternative models, Figure 3.22 for peptide T30 and in Figure 3.23 for peptide T36.
Alternative models represent MS/MS spectra that can support more than one unique phosphorylation pattern.
Figure 3.20:Phosphopeptides identified for the CDeT11–24 protein ofC. plantagineum. Phosphorylated peptides are indicated in red.
Table 3.5: Identification of phosphopeptides of CDeT11–24 by mass spectrometry (MS). Underlined sites represent models that best explain the majority of the tandem mass spectrometry (MS/MS) spectra collected for a given mass-to-charge ratio (m/z). Alternative and equivocal phosphorylation sites for a peptide are marked by letters.
Peptide (start – end) Observed Mr (exp) Mr (calc) Delta Score Sequence
T15A (149 - 160) 686.36 1370.71 1370.60 0.12 28 K.ESDVDSLTQGLK.G T15B (149 - 160) 686.36 1370.71 1370.60 0.12 25 K.ESDVDSLTQGLK.G T15C (149 - 160) 686.36 1370.71 1370.60 0.12 13 K.ESDVDSLTQGLK.G
T30 (338 - 343) 417.72 833.42 833.32 0.10 16 K.DYLSEK.L
T36 (392 - 403) 555.31 1108.60 1108.53 0.08 21 K.GAVGSLIGGGNK.S
Figure 3.21:Fragmentation spectra of the peptide T15 with the three different alternative models.
Figure 3.22:Fragmentation spectrum of the peptide T30.
Figure 3.23:Fragmentation spectrum of the peptide T36.
Lindernia brevidens 11–24 Phosphopeptide Enrichment and Phosphorylation Sites Identification
Lb11–24 from L. brevidens dried leaves was immunochromatographically purified using an IgG-coupled affinity column. Affinity-purified Lb11–24 was digested with trypsin and subjected to phosphopeptide enrichment on TiO2 columns. Nano–LC–MS/MS analysis of phosphopeptide-enriched tryptic digests yielded seven phosphopeptides (Figure3.24) with mass-to-charge ratios (m/z) of 725.38 (2+), 764.68 (2+), 649.23 (3+), 946.82 (2+), 708.75 (3+), 683.92 (2+) and 795.04 (3+) corresponding to the phosphorylated forms of tryptic peptides T12, T14, T15, T33-34, T38-39, T40 and T42 (Table 3.6). Alternative and equivocal phosphorylation sites are marked by letters.
Fragmentation spectra are depicted in Figure3.25for peptide T12, Figure3.26for peptide T14, Figure3.27 for peptide T15, Figure 3.28for peptide T33-34, Figure3.29 for peptide T38-39 and its two alternative models, Figure3.30for peptide T40 and its three alternative models and Figure3.31 for peptide T42 and its five alternative models.
Figure 3.24:Phosphopeptides identified for the Lb11–24 protein ofL. brevidens. Phosphorylated peptides are indicated in red.
Table 3.6: Identification of phosphopeptides of Lb11–24 by mass spectrometry (MS). Underlined sites represent models that best explain the majority of the tandem mass spectrometry (MS/MS) spectra collected for a given mass-to-charge ratio (m/z). Alternative and equivocal phosphorylation sites for a peptide are marked by letters.
Peptide (start - end) Observed Mr (exp) Mr (calc) Delta Score Sequence
T12 (95 - 108) 725.38 1448.75 1448.59 0.16 12 K.GDGGVDTPPQAAER.E T14 (115 - 126) 764.68 1527.35 1526.60 0.75 2 K.HEDVSPTDYQTR.D T15A (127 - 143) 649.23 1944.67 1944.72 −0.04 3
R.DVDSSGQDVGSLTQG-LK.D
T15B (127 - 143) 649.23 1944.67 1944.72 −0.04 3 R.DVDSSGQDVGSLTQG-LK.D
T33-34 (334 - 351) 946.82 1891.82 1890.75 0.87 3 R.VRGTGAGEQTQGGDE-AAK.G
T38-39A (371 - 389) 708.75 2123.23 2123.05 0.17 11 K.AITEKLQLTGNKPT-ADESK.A
T38-39B (371 - 389) 708.75 2123.23 2123.05 0.17 10 K.AITEKLQLTGNKPT-ADESK.A
T40A (390 - 403) 683.92 1365.82 1365.65 0.17 28 K.AATEASPGVVGSIK.G T40B (390 - 403) 683.92 1365.82 1365.65 0.17 31 K.AATEASPGVVGSIK.G T40C (390 - 403) 683.92 1365.82 1365.65 0.17 23 K.AATEASPGVVGSIK.G T42A (414 - 437) 795.04 2382.11 2382.01 0.10 16
K.SNNGSESAGGEQPQS-LGSEGIAAR.E
T42B (414 - 437) 795.04 2382.11 2382.01 0.10 20 K.SNNGSESAGGEQPQS-LGSEGIAAR.E
T42C (414 - 437) 795.04 2382.11 2382.01 0.10 20 K.SNNGSESAGGEQPQS-LGSEGIAAR.E
T42D (414 - 437) 795.04 2382.11 2382.01 0.10 18 K.SNNGSESAGGEQPQS-LGSEGIAAR.E
T42E (414 - 437) 795.04 2382.11 2382.01 0.10 14 K.SNNGSESAGGEQPQS-LGSEGIAAR.E
Figure 3.25:Fragmentation spectrum of the peptide T12.
Figure 3.26:Fragmentation spectrum of the peptide T14.
Figure 3.27:Fragmentation spectrum of the peptide T15.
Figure 3.28:Fragmentation spectrum of the peptide T33-34.
Figure 3.29: Fragmentation spectra of the peptide T38-39 with the two different alternative models.
Figure 3.30:Fragmentation spectra of the peptide T40 with the three different alternative models.
Figure 3.31:Fragmentation spectra of the peptide T42 with the five different alternative models.
Lindernia subracemosa 11–24 Phosphopeptide Enrichment and Phosphorylation Sites Identification
Ls11–24 fromL. subracemosa dried leaves was immunochromatographically purified using an IgG-coupled affinity column. Affinity-purified Ls11–24 was digested with trypsin and subjected to phosphopeptide enrichment on TiO2 columns. Nano–LC–MS/MS analysis of phosphopeptide-enriched tryptic digests yielded seven phosphopeptides (Figure 3.32) with mass-to-charge ratios (m/z) of 543.27 (2+), 632.35 (3+), 585.77 (2+), 873.84 (2+), 491.78 (3+), corresponding to the phosphorylated forms of tryptic peptides T23, T27, T30, T35-36, T48 (Table3.7). Alternative and equivocal phosphorylation sites are marked by letters. Fragmentation spectra are depicted in Figure3.33 for peptide T23 and its two different alternative models, Figure3.34 for peptide T27, Figure 3.35 for peptide T30 and its two different alternative models, Figure 3.36 for peptide T35-36 and its two different alternative models and Figure3.37 for peptide T48.
Figure 3.32: Phosphopeptides identified for the Ls11–24 protein of L. subracemosa. Phosphorylated peptides are indicated in red.
Figure 3.33: Fragmentation spectra of the peptide T23 with the two different alternative models.
Table 3.7: Identification of phosphopeptides of Ls11–24 by mass spectrometry (MS). Underlined sites represent models that best explain the majority of the tandem mass spectrometry (MS/MS) spectra collected for a given mass-to-charge ratio (m/z). Alternative and equivocal phosphorylation sites for a peptide are marked by letters.
Peptide (start - end) Observed Mr (exp) Mr (calc) Delta Score Sequence
T23A (177 - 186) 543.27 1084.52 1084.44 0.07 32 R.DVDSTGQGVK.E T23B (177 - 186) 543.27 1084.52 1084.44 0.07 24 R.DVDSTGQGVK.E T27 (234 - 249) 632.35 1894.01 1893.89 0.13 9
K.ESLLPQSHPLPQ-DEPK.R
T30A (268 - 278) 585.77 1169.53 1168.57 0.96 4 K.ISSASAVIVDK.A T30B (268 - 278) 585.77 1169.53 1168.57 0.96 4 K.ISSASAVIVDK.A T35-36A (319 - 335) 873.84 1745.66 1744.79 0.87 4
K.NMVAEKVSGAGAAV-MSK.V
T35-36B (319 - 335) 873.84 1745.66 1744.79 0.87 4 K.NMVAEKVSGAGAAV-MSK.V
T48 (413 - 421) 491.78 981.55 981.47 0.08 47 K.GVVGSWLGK.N
Figure 3.34:Fragmentation spectrum of the peptide T27.
Figure 3.35: Fragmentation spectra of the peptide T30 with the two different alternative models.
Figure 3.36: Fragmentation spectra of the peptide T35-36 with the two different alternative models.
Figure 3.37:Fragmentation spectrum of the peptide T48.
Sequence Comparison among C. plantagineum, L. brevidens and L. subracemosa 11–24 Sequences and their Phosphorylation Sites.
C.plantagineum MESQLHRPTEQEV---MEGQTADHGEKKSMLAKVKEKAKKLKGSINKKHGSSQD 51 L.subracemosa MESQMHRPSEQEHPQHVASDNEMVEDQAEHGEKKSMLKKVKDKAKKIKGTI-KKHGLGQD 59 L.brevidens MESQMHRPVEQET---VEGQGEQGGKMSVLKKVKEKAKKLKGSIKKHGSNSGQ 50
****:*** *** .::* * *:* ***:****:**:* *: . . : C.plantagineum DDADNDEEINTSPAVHG-APGTSP---PPPTQG--- 80 L.subracemosa Q---EDEEIKTNPAVHGPPPGRVPPVITGTNPPPMQGGFNLEKPTDTREDRYDHHSDNVK 116 L.brevidens EEDGSDEEMDTSPAVHG-APGMTP---PPTQGG---DLEKP 84
: .***:.*.***** .** * ** **
C.plantagineum ---GEYGGLS-ERDVNIPHPLASTQANLDKPADVTDASRELQVPPPVPETTPEVSDKGLT 136 L.subracemosa GGGGEYTSLR-EKGVTSPPQDMETEFNFPKPEKNEPEMKNITKPDVKTETSDITPSDYQT 175 L.brevidens ADAGGFTSLKGDGGVDTPPQAAEREFNFPKHEDVS---PTDYQT 125
* : .* : .* * . : *: * . . . *
C.plantagineum EDLGSNAGQGVKESDVDSLTQGLKGVNYGGDDSNPLSGQEHQTISD---EPKSLPGQ 190 L.subracemosa RDVDS-TGQGVKEADVGSLTQGLKKVSVG-DESKPLPGEEQPPSYSGSHGQFAPQSTPTK 233 L.brevidens RDVDS-SGQ---DVGSLTQGLKDMNVGGDESKAVPEVQEQPRST---PAAAE 170
.*:.* :** **.******* :. * *:*:.:. :. . . . : C.plantagineum GNDLPQSHPS-SEDEPKKFDANDQPQSMPQDT-ITGKLSSVPAVIIDRAAAAKNVVASKL 248 L.subracemosa ESLLPQSHPL-PQDEPKRYDPN-HPDSMPQDT-ITGKISSASAVIVDKAATAKNVVASKL 290 L.brevidens ETHLPQSHPVPAEDEPKKYDPN-RPDSTPQDTTYIGKITSVPAVIVDKAAAAKNVVASKL 229
. ****** .:****::*.* :*:* **** **::*..***:*:**:*********
C.plantagineum GYGGS---QAQESAADA 262 L.subracemosa GYGG---QTQESSD-- 301 L.brevidens GYGANNQAQEPTTTPDVVGGGGAATQQKKPLTETAAEYKNMVAEKLGYGASKAQESVDVG 289
***. ::***
C.plantagineum ---GAAQQKKPLTETAAEYKNLVAEKLTPVYEKVAGAGSTVTSKVWGSGGTTAGEQTQGG 319 L.subracemosa ---GGAQQKKPLTETAAEYKNMVAEKVS---GAGAAVMSKVRGSG---TGEQAQGG 348 L.brevidens GDGGATQQKKPLTETAAEYKNMVAEKLAPVYGKVSGAGTGVISRVRGTG---AGEQTQGG 346
*.:***************:****:: ***: * *:* *:* :***:***
C.plantagineum EGVVDGGGAASNKGVFTKDYLSEKLKPGDEDKALSQAIMEKLQLSKKPAVEGGAGDETKA 379 L.subracemosa ---GGGDEASKGVSTKDYLSDKLKPGEEDKALSKAITEKLQVGKNEPRDG----SKAA 399 L.brevidens ---DEAAKG----SYLSEKLKPGDEDKALSKAITEKLQLTGNKPTAD---ESKAA 391
: ** .***:*****:******:** ****: : . . .. * C.plantagineum SESSPG-VVGTIKGAVGSLIGGGNKSSGAESAA---AADEQTQALGE---- 422 L.subracemosa NESSPGGVVSSLKGVVGSWLG---KNNGSEEAA---AAREEANAERKV-EQ 443 L.brevidens TEASPG-VVGSIKGVVGSLLGG-KSNNGSESAGGEQPQSLGSEGIAAREGADVVEPTAEQ 449
.*:*** **.::**.*** :* ...*:*.*. ** * ::. **
The amino acid sequence alignment of the three sequences is shown: the identified phosphorylation sites are underlined. “*”:identical. “:”:conserved substitutions. “.”:semi-conserved substitutions.