Chinese Journal of Chromatography ›› 2021, Vol. 39 ›› Issue (1): 77-86.DOI: 10.3724/SP.J.1123.2020.07041
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ZHANG Baohui, WANG Chentong, GUO Miao, XIAO Hua*()
Received:
2020-07-31
Online:
2021-01-08
Published:
2020-12-20
Contact:
XIAO Hua
Supported by:
CLC Number:
ZHANG Baohui, WANG Chentong, GUO Miao, XIAO Hua. Affinity chromatography based phosphoproteome research on lung cancer cells and its application[J]. Chinese Journal of Chromatography, 2021, 39(1): 77-86.
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URL: https://www.chrom-china.com/EN/10.3724/SP.J.1123.2020.07041
Enrichment method | Protein initiation amount/μg | Number of identified phosphopeptides | Number of identified peptides | Percent of phosphopeptides/ % | Phosphorylation PSM | PSM | Percent of phosphorylation PSM/% | Number of phosphorylation sites |
---|---|---|---|---|---|---|---|---|
PE | 200 | 21 | 40 | 52.50 | 53 | 75 | 70.67 | 31 |
400 | 50 | 114 | 43.86 | 197 | 333 | 59.16 | 88 | |
Vortexing | 200 | 31 | 65 | 47.69 | 55 | 96 | 57.29 | 43 |
400 | 62 | 112 | 55.36 | 192 | 272 | 70.59 | 88 |
Table 1 Specificity of phosphopeptide enrichment by Ti4+-IMAC-S using SPE and vortexing methods
Enrichment method | Protein initiation amount/μg | Number of identified phosphopeptides | Number of identified peptides | Percent of phosphopeptides/ % | Phosphorylation PSM | PSM | Percent of phosphorylation PSM/% | Number of phosphorylation sites |
---|---|---|---|---|---|---|---|---|
PE | 200 | 21 | 40 | 52.50 | 53 | 75 | 70.67 | 31 |
400 | 50 | 114 | 43.86 | 197 | 333 | 59.16 | 88 | |
Vortexing | 200 | 31 | 65 | 47.69 | 55 | 96 | 57.29 | 43 |
400 | 62 | 112 | 55.36 | 192 | 272 | 70.59 | 88 |
Material | Number of identified phosphopeptides | Number of identified peptides | Percent of phosphopeptides/ % | Phosphorylation PSM | PSM | Percent of phosphorylation PSM/% | Number of phosphorylation sites | Number of phosphoproteins |
---|---|---|---|---|---|---|---|---|
Ti4+-IMAC-S | 1644 | 2258 | 72.81 | 3297 | 4016 | 82.10 | 1804 | 914 |
Ti4+-IMAC-L | 1002 | 2072 | 48.36 | 2006 | 3119 | 64.32 | 1095 | 626 |
Table 2 Specificity of phosphopeptide enrichment for 95C cell line by Ti4+-IMAC-S and Ti4+-IMAC-L
Material | Number of identified phosphopeptides | Number of identified peptides | Percent of phosphopeptides/ % | Phosphorylation PSM | PSM | Percent of phosphorylation PSM/% | Number of phosphorylation sites | Number of phosphoproteins |
---|---|---|---|---|---|---|---|---|
Ti4+-IMAC-S | 1644 | 2258 | 72.81 | 3297 | 4016 | 82.10 | 1804 | 914 |
Ti4+-IMAC-L | 1002 | 2072 | 48.36 | 2006 | 3119 | 64.32 | 1095 | 626 |
Cell line | Phosphosite number per protein | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | ||
MRC5 | 331 | 88 | 30 | 15 | 8 | 2 | 1 | 1 | 0 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | |
95C | 502 | 169 | 59 | 21 | 12 | 13 | 7 | 2 | 2 | 0 | 0 | 1 | 2 | 0 | 0 | 0 | |
95D | 658 | 190 | 90 | 42 | 27 | 11 | 14 | 4 | 5 | 3 | 1 | 0 | 3 | 3 | 2 | 0 |
Table 3 Frequency of phosphosite number per protein
Cell line | Phosphosite number per protein | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | ||
MRC5 | 331 | 88 | 30 | 15 | 8 | 2 | 1 | 1 | 0 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | |
95C | 502 | 169 | 59 | 21 | 12 | 13 | 7 | 2 | 2 | 0 | 0 | 1 | 2 | 0 | 0 | 0 | |
95D | 658 | 190 | 90 | 42 | 27 | 11 | 14 | 4 | 5 | 3 | 1 | 0 | 3 | 3 | 2 | 0 |
Fig. 3 Numbers and ratios of (a) mono- and multi-phosphopeptides and (b) different phosphosites1-P: singly phosphorylation; 2-P: doubly phosphorylation; 3-P: triply phosphorylation.
GO catalogue | Term | Gene percent/% | False discovery rate |
---|---|---|---|
Biological process | mRNA metabolic process | 14.84 | 2.20E-29 |
RNA processing | 12.97 | 7.98E-28 | |
mRNA processing | 16.67 | 5.06E-25 | |
nucleic acid metabolic process | 6.50 | 5.58E-25 | |
RNA splicing | 17.90 | 1.10E-24 | |
nucleobase-containing compound metabolic process | 6.13 | 2.34E-24 | |
organelle organization | 6.90 | 3.37E-23 | |
cellular component organization or biogenesis | 5.73 | 5.65E-23 | |
regulation of metabolic process | 5.33 | 4.28E-22 | |
regulation of organelle organization | 9.87 | 2.77E-21 | |
Cell component | nuclear part | 7.89 | 5.86E-59 |
nuclear lumen | 8.04 | 4.66E-56 | |
nucleoplasm | 8.39 | 1.72E-51 | |
intracellular part | 4.43 | 1.78E-50 | |
nucleus | 6.11 | 2.14E-48 | |
intracellular | 4.35 | 1.11E-47 | |
intracellular organelle | 4.72 | 1.77E-46 | |
organelle | 4.66 | 2.10E-45 | |
cytosol | 6.84 | 3.44E-44 | |
intracellular non-membrane-bounded organelle | 7.37 | 3.45E-42 | |
Molecular function | protein binding | 5.45 | 2.23E-25 |
binding | 4.39 | 5.64E-23 | |
cytoskeletal protein binding | 10.09 | 9.23E-17 | |
RNA binding | 10.00 | 8.07E-16 | |
enzyme binding | 6.92 | 1.94E-15 | |
heterocyclic compound binding | 5.07 | 2.09E-12 | |
nucleic acid binding | 5.70 | 5.80E-12 | |
protein domain specific binding | 9.63 | 6.60E-12 | |
organic cyclic compound binding | 5.00 | 8.06E-12 | |
kinase binding | 9.44 | 7.03E-11 |
Table 4 Gene ontology (GO) analysis of the phosphoproteins corresponding to dysregulated phosphorylation sites
GO catalogue | Term | Gene percent/% | False discovery rate |
---|---|---|---|
Biological process | mRNA metabolic process | 14.84 | 2.20E-29 |
RNA processing | 12.97 | 7.98E-28 | |
mRNA processing | 16.67 | 5.06E-25 | |
nucleic acid metabolic process | 6.50 | 5.58E-25 | |
RNA splicing | 17.90 | 1.10E-24 | |
nucleobase-containing compound metabolic process | 6.13 | 2.34E-24 | |
organelle organization | 6.90 | 3.37E-23 | |
cellular component organization or biogenesis | 5.73 | 5.65E-23 | |
regulation of metabolic process | 5.33 | 4.28E-22 | |
regulation of organelle organization | 9.87 | 2.77E-21 | |
Cell component | nuclear part | 7.89 | 5.86E-59 |
nuclear lumen | 8.04 | 4.66E-56 | |
nucleoplasm | 8.39 | 1.72E-51 | |
intracellular part | 4.43 | 1.78E-50 | |
nucleus | 6.11 | 2.14E-48 | |
intracellular | 4.35 | 1.11E-47 | |
intracellular organelle | 4.72 | 1.77E-46 | |
organelle | 4.66 | 2.10E-45 | |
cytosol | 6.84 | 3.44E-44 | |
intracellular non-membrane-bounded organelle | 7.37 | 3.45E-42 | |
Molecular function | protein binding | 5.45 | 2.23E-25 |
binding | 4.39 | 5.64E-23 | |
cytoskeletal protein binding | 10.09 | 9.23E-17 | |
RNA binding | 10.00 | 8.07E-16 | |
enzyme binding | 6.92 | 1.94E-15 | |
heterocyclic compound binding | 5.07 | 2.09E-12 | |
nucleic acid binding | 5.70 | 5.80E-12 | |
protein domain specific binding | 9.63 | 6.60E-12 | |
organic cyclic compound binding | 5.00 | 8.06E-12 | |
kinase binding | 9.44 | 7.03E-11 |
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