Mass Spectrometric Analysis of Human Transferrin in Different Body Fluids

Abstract

In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.

The impact of mass spectrometry in the diagnosis of congenital disorders of glycosylation

“…The observed mass differences (Δ =1,322 Da) with respect to the fully glycosylated Tf, reflected the occurrence of truncated N-glycans both lacking the entire arm connected through the Man (alfa1-3) linkage (Kleinert et al 2003).…”

“…In order to make more feasible the use of MS to laboratory diagnosis of CDG, liquid chromatography (LC) techniques were coupled with ESI MS for speeding-up sample preparation and analysis time (Kleinert et al 2003). In particular, since 2000, the Mayo Clinic laboratories adopted an on-line immunoaffinity liquid chromatography mass spectrometry approach (LC-MS) as clinical testing for CDG Bergen et al 2001).…”

Carbohydrate-deficient transferrin (CDT) as a marker of alcohol abuse: A critical review of the literature 2001–2005

“…In a study performed on 152 blood samples drawn from 38 persons (four tubes for each person), divided into four groups on the basis of different time intervals between blood sample collection and centrifugation (1,24,48 and 144 h, respectively), the means and medians of CDT were reported to increase with the time of whole blood storage. ANOVA analysis of between-group differences was significant for mean CDT concentrations between 1 and 144 h of whole blood storage [20].…”

The potential of mass spectrometry to study iron-containing proteins used in clinical diagnosis

“…different content in sialic acids, mannose, fucose, etc.). The glycans differ in their degree of branching showing bi, tri and tetra-antennary structures, each antenna terminated in a negatively charged sialic acid (N-acetylneuraminic acid) molecule [20]. Therefore, Tf presents different microheterogeneity due both, to its final iron content and to the composition of the N-glycan chains.…”

“…This methodology was slightly modified [83] for Tf isoforms in blood, by using a blood spot cartridge before the immunoaffinity column, and allowed Tf glycoforms characterization in whole blood, serum or plasma from chronic alcoholic and CDG patients. Also, Kleinert et al [20] published a procedure which did not require Tf immunoextraction of Tf isoforms from serum, plasma and CSF. That method was based on three steps: a) Tf isolation by anion exchange (AE) HPLC with UV detection at 254 nm; b) preconcentration of Tf fraction; c) detection of Tf isoforms by RPLC-ESI-MS. Up to 10 different Tf isoforms in serum from a CDG patient were identified, according to their molecular masses.…”