Jane Y. Yang(1), David A. Herold(1), Mark W. Duncan(2), Stephen J. Hattan(3), Kenneth C. Parker(3), Marvin L. Vestal(3)
(1)University of California San Diego, (2)University of Colorado School of Medicine, (3)SimulTof Systems
Percent HbA1c, the standard measure of glycated hemoglobin used to diagnose and monitor diabetes mellitus, correlates linearly with total glycated hemoglobin (tGHb). MALDI-TOF mass spectrometry of whole blood hemolysates allows the direct quantitation of the total glycated hemoglobin (tGHb) ratios of alpha and beta chains from a single mass spectrum. Sample preparation for this approach is minimal, analysis is rapid, 80 spots in 20 min (15 sec/spot), and hemoglobin variants are also detected. tGHb by MALDI is reproducible with intra-plate CVs < 1.4%, linear vs. cation exchange HPLC (y = 1.20x – 1.07; R2 = 0.99) from 2.7% to 22% A1c. MALDI method is faster and less expensive than HPLC.
Hemoglobin A1c (HbA1c), reported as the percent of hemoglobin (Hb) glycated on the N-terminal valine of the β-chain of total β-chain, is the standard measure of glycated Hb utilized in the diagnosis and monitoring of diabetes mellitus. Reference guidelines for non-diabetic/normal HbA1c is <6%, compared to near normoglycemic 6-7% or uncontrolled diabetes >9%. Glycated hemoglobin reflects a weighted average blood glucose concentration over the life span of a red blood cell, typically 120 days. However, glycation may occur at any of the amino groups within hemoglobin.
Methods used to determine HbA1c include cation-exchange HPLC, gel electrophoresis, immunoassay, and boronate affinity chromatography. These methods have intensive sample preparation, yield spurious results with hemoglobin variants, and take 2 minutes or more per sample. Boronate affinity chromatography determines the percent total glycated Hb (tGHb) for the β-subunit, which linearly correlates to HbA1c percentages.
We demonstrate a new approach to determine HbA1c from tGHb by quantitative MALDI-TOF mass spectrometry (MS) of whole blood hemolysates. MALDI-TOF MS allows the direct detection of unmodified Hb chains and corresponding glycated forms from a single acquisition/mass spectrum. Additionally, sample preparation consists of simple dilutions and batched data acquisition is fast.
We assayed intra- and inter-plate variability, linearity, and patient samples. Our MS based analysis can also capture Hb variants and α-chain glycation, which enhances spectral quality control.
Hb / HbA1c Standard Curve preparation. Lyphochek® Hemoglobin A1c Linearity Set (BioRad, Hercules, CA) level 6 standard (16-22% HbA1c) was separated following the A1c analytical protocol using a Mono S cation-exchange column (GE Healthcare, Uppsala, Sweden). Lyophilized hemoglobin standard (Lee Biosolutions, St. Louis, MO) was weighed, reconstituted, and diluted to create a range of protein concentrations; triplicate absorbance measurements at each concentration were averaged and used to construct an absorbance based calibration curve. Separated Hb and HbA1c from the BioRad A1c calibration standard were reanalyzed by absorbance against our calibration curve for protein concentration determination. Isolated Hb and HbA1c were then remixed in proportions that mimic and span clinically relevant HbA1c blood levels (1.36-20%). Constructed standards were then analyzed by MALDI MS, as described below, to create an MS based calibration curve.
MALDI sample preparation. Specimens, either standards or whole blood collected in EDTA tubes following normal phlebotomic protocol, were diluted 1:1000 in DI H2O, vortexed, and centrifuged (12000 rpm for 5 min). The resulting hemolysate was mixed 1:1 with 10 mg/mL sinapinic acid (50% ACN, 0.1% TFA) and 1 µL of this preparation was spotted onto a stainless steel MALDI target. For the purposes of assay evaluation and development, all samples were analyzed in replicates of 5.
MALDI-TOF MS. Mass spectra were acquired on a SimulTOF 100 MALDI-TOF mass spectrometer (SimulTOF Systems, Sudbury, MA) in linear mode using positive ion polarization. Individual spectra were an average of 100 laser shots over 5000-20,000 Da mass range using the following acquisition parameters: acceleration voltage 20 kV, focus mass 15000, laser pulse frequency 1000 Hz, laser pulse energy 9 µJ, scan rate 1 mm/min at 100 µm raster over each sample spot.
Data Analysis/Calculation of HbA1c. Post-acquisition data processing was performed by averaging all spectra that passed a 20 mV minimum signal intensity threshold at each sample position. These spot-averaged spectra were calibrated using average mass values of M1+ and M2+ of Hb β- (15868.51, 7934.75 Da) and α- subunits (15127.74, 7564.37 Da). Peak areas for unmodified and glycated (+162 Da) β- and α-subunits were estimated using an algorithm developed in-house (SimulTOF Systems). Percent ratios of the tGHb for each subunit were calculated using the peak areas in the following equation [glycated subunit / (unmodified + glycated subunit)]*100.
MALDI-TOF MS of A1c yielded intra-plate CV < 0.9%. The IFCC primary reference material preparation was mimicked to generated a standard curve for MALDI MS. HbA1c by MALDI was linear from 1.7-15.2% (R2 = 0.99) with CVs < 4%. The set of patient samples was prepared in replicates of 5 per sample per plate in replicates of 3 plates (15 spots per sample). MALDI MS results compared to the corresponding HPLC-determined values were linear from 4.9 – 9.9% A1c (y = 0.99x + 0.12; R2 = 0.996) with intra-plate CV ≤ 3.8% and inter-plate CV < 7%.
MALDI MS does not rely on chromatographic retention times, and thus allows simultaneous detection of unmodified and glycated α- and β-chains of hemoglobin and hemoglobin variants. Glycation on the N-terminal valine changes the overall charge and mass of the β-chain. Based on this charge difference, cation exchange HPLC separates HbA1c from Hb, and the absorbance of heme is used for quantification. Hb modified elsewhere and hemoglobin variants are detectable by absorbance, but do not elute during the expected times, compromising correct quantification of the Hb and HbA1c peaks.
Covalent attachment of glucose to hemoglobin adds 162 Da/glucose to the mass of the hemoglobin chain. From the mass spectrum, the integrated peak areas (ion counts) for the glycated hemoglobin chain and unmodified hemoglobin chain can be accurately quantified to obtain a tGHb ratio, which is then used to calculate HbA1c. In addition to indirect determination of HbA1c, MALDI MS detects glycation of the α-chain and doubly charged Hb molecules. This additional quantitative information can corroborate the glycation ratios, detect variant Hb, and indicate quality of spectra. We have found that the ratio of glycated α chain to total α chain tracks closely with the β-chain ratio if no variants are present. For hemoglobin variants such as hemoglobin S, the α- and β-chain glycation ratios will be inconsistent. Therefore, these two ratios can be used to rule out variants or spectra of low quality.
If the quality of the spectra is poor, based on inconsistent α-, β-, doubly charged α-, and doubly charged β-chain glycation ratios, more spectra can be collected from the same sample within minutes, or if necessary, from duplicate samples.
The high inter-plate CV was due to inconsistent matrix crystallization, typically of one spot out of the 5 replicates. Poor matrix crystallization resulted in decreased signal intensity, and was evaluated by visual inspection of the MALDI target plate and by spectra quality. Many factors contribute to the consistency of matrix crystallization, which needs to be optimized to decrease the inter-plate CV.
With optimization, HbA1c from tGHb by MALDI-TOF MS is an approach that will be useful in the clinical laboratory.