Transferrin(TF) and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid(asialo-, monosialo-, and disialo-transferrin) and asialo-TF as well as disialo-TF, with one or several oligosaccharides released in human serum transferrin(hTf). Here, hTf has been purified by native gradient polyacrylamide gel electrophoresis(PAGENG) before use. The hTf extracted with the electron-transfer approach showed a single subunit band(77.1 Da) in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing(IEF) gels, namely, hTf-2Fe3+ and apo-hTf, without finding any other transferrin isoforms. A reversed phase HPLC(RP-HPLC) equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting(PMF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and database search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation. The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe3+ and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as, carbohydrate-deficient transferrin(CDT). Transferrin (TF) and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid (asialo-, monosialo-, and disialo- transferrin) and asialo-TF as well as disialo- TF, with one or several oligosaccharides released in human serum transferrin (hTf). Here, hTf has been purified by native gradient polyacrylamide gel electrophoresis (PAGENG) before use. The hTf extracted with the electron-transfer approach showed a single subunit band (77.1 Da) in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing (IEF) gels, namely, hTf-2Fe3 + and apo- hTf, without finding any other transferrin isoforms. HPLC) equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting (PMF), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and datab ase search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation. The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe3 + and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as, carbohydrate-deficient transferrin (CDT).