Sample preparation of blood plasma enables baseline separation of iron metalloproteins by SEC-GFAAS

Abstract

The analysis of human plasma for biomarkers holds promise to revolutionize disease diagnosis, but is hampered by the inherent complexity of the plasma proteome. One way to overcome this problem is to analyze plasma for a sub-proteome, such as the metalloproteome. Previous studies employing size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) have revealed that plasma contains ~12 copper, iron and zinc metalloproteins. This included the iron metalloproteins transferrin (Tf) and a recently identified haptoglobin-hemoglobin (Hp-Hb) complex, which is formed in plasma when red blood cells rupture. Since this SEC-ICP-AES method required a sample volume of 500 µL to generate diagnostically useful results, we sought to develop an alternative SEC-based hyphenated approach using a smaller SEC column (150 x 5 mm I.D.) and a graphite furnace atomic absorption spectrometer (GFAAS) as the iron-specific detector. A designed interface enabled the integration of the SEC system with the GFAAS. Baseline separation between the Hp-Hb complex and Tf was achieved by developing a sample preparation procedure which involved the chelating agent-based mobilization of Fe from Tf to a small molecular weight Fe complex. Spiking of human plasma (1.0 mL) with red blood cell lysate (1-2 µL) increased only the intensity of the Fe peak corresponding to the Hp-Hb complex, but not that of Tf. Since the developed SEC-GFAAS method requires only 50 µL for analysis, it can now be employed for the cost-effective quantification of the clinically relevant Hb-Hp complex in human plasma in <50 min.

Recent Advances in Metalloproteomics

“…For metal quantitation, the overwhelming majority of LC-and CE-based studies involve ICP-MS [11,25,27], with a few examples for Inductively-Coupled Plasma Optical Emission Spectroscopy (ICP-OES) [58] and Graphite Furnace Atomic Absorption Spectrometry (GFAAS) [59]. For metal quantitation for 2D-GE, at least four methods are available: Laser-ablation ICP-MS (LA-ICP-MS) [60], X-ray fluorescence (XRF) [61][62][63], application of a metallochromic dye or fluorescent sensor [64], and autoradiography of proteins labelled with a radioactive metal ion.…”

Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age

“…Considering the emerging role that red blood cells may play in certain disease processes [ 44 ] and given the conceptual link between the dominant Hp isoform in serum and certain neurodegenerative processes [ 36 , 40 , 42 , 45 ], the analysis of plasma/serum from patients with distinct neurodegenerative diseases has the potential to uncover novel metal-based biomarkers. The inherent potential that the analysis of plasma/serum for iron metalloproteins may offer to facilitate disease diagnosis is complemented by the development of analytical methods which not only require much smaller volumes for metalloprotein analysis (50 μL versus 500 μL in this study), but also uses equipment that could be operated by technicians in clinical biochemistry laboratories [ 7 ].…”

Recent Progress in Coordination Chemistry

“…Based on the well-established metabolism of cisplatin in blood plasma [76], however, it is unknown if the integrity of the RBC lipid bilayer membrane is perturbed by cisplatin or its hydrolysis products. A recently developed metallomics tool can be employed to analyze blood plasma (50 µL) for a hemoglobin-haptoglobin complex to estimate the RBC rupture that is induced by a pharmacologically relevant dose of a metallodrug when added to whole blood [132].…”