In-depth Phosphoproteome Analysis from Microscale Samples with Rapid In-situ One-tip IMAC Strategy

doi:10.1007/s40242-026-5299-6

Abstract

Abstract: Compared to advancements in microscale sample proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We developed a simple and rapid in-situ one-tip immobilized metal affinity chromatography (IMAC) phosphoproteomics workflow integrated with data-dependent acquisition without exclusion mass spectrometry for microscale phosphoproteomic analysis. The phosphopeptide enrichment material, made from hydrophilic cotton fibers with immobilized titanium ions, is easy to pack into a pipette tip. It enables fast, efficient enrichment through aspiration and dispensing, minimizing sample loss and enhancing sensitivity. Ti-cotton tip successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity [bovine serum albumin (BSA)/standard phosphopeptide=2000:1)], and enabled 10332 phosphosites assigned to 1992 phosphoproteins from 20000 HeLa cells. Furthermore, a large scale of 36521 phosphosites assigned to 4452 phosphoproteins with 8952 new sites could be identified by using 30 μg of HeLa cell lysates as starting material via combination with high-pH reversed-phase (RP) fractionation. Taken together, our strategy is promising to serve as a widely applicable and robust platform for achieving large-scale and highly sensitive phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex microscale biological systems.

Key words: Phosphoproteomics, Microscale analysis, Immobilized metal affinity chromatography, Sensitivity, Phosphopeptide enrichment

LIN Yao, WANG Xinyu, WANG Xinyue, LUO Mingyue, YANG Mei, WU Ci. In-depth Phosphoproteome Analysis from Microscale Samples with Rapid In-situ One-tip IMAC Strategy[J]. Chemical Research in Chinese Universities, 2026, 42(2): 504-513.

References

[1] Zhang J., Chen M., Jiang H., Sun H., Ren J., Yang X., Liu S., Wang D., Huang Z., Liu J., Adv. Sci., 2024, 11, 2403120.
[2] Zhang Z., Yuan H., Ni R., Yin J., Li M., Yang P., Cao X., Zhou J., Su X., Chen Y., Talanta, 2024, 278, 126487.
[3] Shen C., Huang Z., Chen X., Wang Z., Zhou J., Wang Z., Liu D., Li C., Zhao T., Zhang Y., Biosens. Bioelectron., 2023, 242, 115719.
[4] Khodaparast M., Sharley D., Marshall S., Beddoe T., NPJ Clean Water, 2024, 7, 74.
[5] Xu J., Akhtar M., Meng W., Bai J., Prince S., Huang R., Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol., 2025, 17, e70022.
[6] Majid M., Cheng S., Chem. Res. Chinese Universities, 2025, 41, 1278.
[7] Nie S., Zhang P., Zhang Q., Ding C., Chem. Res. Chinese Universities, 2025, 41, 975.
[8] Lamprou E., Kalligosfyri P. M., Kalogianni D. P., Biosensors, 2025, 15, 68.
[9] Shi L., Chen H., Zhang Z., Wang Y., Ren W., Huang J., Front. Cell. Infect. Microbiol., 2025, 15, 1681779.
[10] Shi C., Yu Z., Tan H., Li W., Wang Y., Wang Y., Zhang Q., Man Y., ACS Sens., 2025, 10, 9108.
[11] Rolando J. C., Jue E., Barlow J. T., Ismagilov R. F., Nucleic Acids Res., 2020, 48, e42.
[12] Guo W., Jiang M., Xie Y., Xu H., Sun Z., Biosensors, 2025, 15, 717.
[13] Wang R., Xu Q., Liu N., Li Y., Sun N., Talanta, 2025, 128636.
[14] Teng G., Lin G., Wei P., Li L., Chen H., Chen Q., Lin Q., Analyst, 2025, 150, 1419.
[15] Zhu Z., Li R., Zhang H., Wang J., Lu Y., Zhang D., Yang L., Biosens. Bioelectron., 2022, 204, 114076.
[16] Dong M., Kshirsagar A., Politza A. J., Guan W., ACS Nano, 2024, 18, 7170.
[17] Gao J., Yang C., Wu X., Huang W., Ye J., Yuan R., Xu W., Sens. Actuators B: Chem., 2023, 380, 133354.
[18] Quyen T. L., Vinayaka A. C., Golabi M., Nguyen T., Ngoc H. V., Bang D. D., Wolff A., ACS Sens., 2022, 7, 3343.
[19] Hardinge P., Murray J. A. H., Sci. Rep., 2020, 10, 916.
[20] Koryukov M. A., Oscorbin I. P., Novikova L. M., Gordukova M. A., Turina I. E., Galeeva E. V., Kudlay D. A., Filipenko M. L., Int. J. Mol. Sci., 2024, 25, 7215.
[21] Lin Q., Gou H., Qu X., Jia K., Deng Y., Li D., Cai Q., Liang Y., Xu X., Li Y., Lin J., Li L., Jiang Y., Du S., Deng L., Yan B., Liu R., Li C., Zhang J., Liao M., Adv. Sci., 2025, 12, 2502708.
[22] Zhang X., Zhao Y., Zhu X., Tian W., Zhang C., Anal. Bioanal. Chem., 2024, 416, 1971.
[23] He S., Zhou Y., Xie Y., Zhang K., He Q., Yin G., Zou H., Hu Q., Zhang S., He H., Wang D., Talanta, 2023, 252, 123823.
[24] Xie Y., Jiang K., Zhang Y., Cao L., Guo X., Shi J., Ding X., Yin K., Anal. Chem., 2024, 96, 15493.
[25] Liu X., Zhang J., Hua K., Cui Y., Talanta, 2025, 285, 127300.
[26] Cai C., Hou X., Huang B., He J., Wu X., He Q., Food Control, 2025, 167, 110809.
[27] Zhang C., Luo Z., Huang T., Nian J., Sun J., Lin X., ACS Food Sci. Technol., 2025, 5, 1042.
[28] Chen Y., Zhu Y., Wang X., Peng C., Wang R., Wang T., Che Y., Wu J., Xu J., Talanta, 2024, 267, 125258.
[29] Li H., Lu H., Tang Y., Wang H., Xiao Y., Li B., Angew. Chem. Int. Ed., 2022, 61, e202209496.
[30] Zhou X., Wu M., Zhou Y., Su F., He Y., Ding J., Xie L., Front. Public Health, 2025, 13, 1493589.
[31] Sripan P., Prouté M., Ngo-Giang-Huong N., Rangdaeng S., Putiyanun C., Halue G., Leenasirimakul P., Thongpaen S., Buranabanjasatean S., Le Coeur S., Cancer Commun., 2025, 45, 525.
[32] Mortaki A., Douligeris A., Daskalaki M.-A., Bikouvaraki E.-S., Louizou E., Daskalakis G., Rodolakis A., Grigoriadis T., Pappa K. I., Biomedicines, 2025, 13, 1139.
[33] Wang M., Liang H., Yan Y., Bian R., Huang W., Zhang X., Nie J., Hum. Vaccin. Immunother., 2024, 20, 2343192.
[34] Siriaunkgul S., Suwiwat S., Settakorn J., Khunamornpong S., Tungsinmunkong K., Boonthum A., Chaisuksunt V., Lekawanvijit S., Srisomboon J., Thorner P. S., Gynecol. Oncol., 2008, 108, 555.
[35] Li N., Cheng C., Liang R., Zhu Q., Xue F., Xu L., Shi Y., Luo H., Yu S., He J., Cancer Epidemiol., 2023, 84, 102360.
[36] Hu Y., Liu L., Wang C., Zhou J., Liu R., Lv Y., Chem. Commun., 2024, 60, 976.
[37] Koster S., Gurumurthy R. K., Kumar N., Prakash P. G., Dhanraj J., Bayer S., Berger H., Kurian S. M., Drabkina M., Mollenkopf H.-J., Nat. Commun., 2022, 13, 1030.
[38] Ghosh A., Sinha A., Ghosh A., Roy S., Mallick S., Kumar V., Mathai S., Bhaumik J., Mukhopadhyay A., Sen S., Hum. Genomics, 2024, 18, 91.
[39] Zhang B., Song Y., Sun S., Han R., Hua C., van der Veen S., Cheng H., J. Virol., 2019, 93, 10.1128/jvi. 00172.