Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

Applications of MALDI Mass Spectrometry in Clinical Chemistry.

BACKGROUND: MALDI-TOF mass spectrometry (MS) is set to make inroads into clinical chemistry because it offers advantages over other analytical platforms. These advantages include low acquisition and operating costs, ease of use, ruggedness, and high throughput. When coupled with innovative front-end strategies and applied to important clinical problems, it can deliver rapid, sensitive, and cost-effective assays. CONTENT: This review describes the general principles of MALDI-TOF MS, highlights the unique features of the platform, and discusses some practical methods based upon it. There is substantial potential for MALDI-TOF MS to make further inroads into clinical chemistry because of the selectivity of mass detection and its ability to independently quantify proteoforms. SUMMARY: MALDI-TOF MS has already transformed the practice of clinical microbiology and this review illustrates how and why it is now set to play an increasingly important role in in vitro diagnostics in particular, and clinical chemistry in general.

Bifunctional glass membrane designed to interface SDS-PAGE separations of proteins with the detection of peptides by mass spectrometry.

We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-PAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.

Novel three-dimensional MALDI plate for interfacing high-capacity LC separations with MALDI-TOF.

Novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) sample plates employing collimated-hole structures have been developed that allow capture and concentration of samples while simultaneously acting as a sink for carrier solvents. These plates were designed to provide an efficient interface between higher-capacity liquid chromatography (LC) separations and MALDI-TOF mass spectrometry (MS). LC-MALDI using conventional plates can accommodate the low-flow (< 1 microL/min) separation protocols typically used in on-line LC-MS methods, and can also be used with higher flow rate, larger columns, but are ultimately limited by the capacity of the two-dimensional surface onto which the sample is deposited. Typically, about 1 microL of chromatographic effluent plus 1 microL of matrix solution can be deposited and dried on a ca. 3 mm diameter spot. Deposition rates (spot dwell time) are determined by the chromatographic resolution and the flow rate. To overcome this limitation, a new three-dimensional MALDI sample plate has been developed using collimated-hole structures (CHS) with monolithic chromatography media filling the holes in the collimated-hole structures. These new plates retain all of the required features of conventional sample plates, commonly formed from stainless steel, but provide additional capacity for capturing and concentrating samples. Results are presented from reversed-phase separation of peptides on a 1 mm i.d. column operating at flow rate of 50 microL/min. Typically, 10 microL of effluent can be collected on a single spot, and sample and matrix dried on a 1 mm diameter spot, to yield about 30-fold enrichment of sample concentration in matrix crystals on the surface compared to the conventional plate. Sample loadings ranging from 1 fmol to 10 pmol/spot were investigated.

Methodology utilizing MS signal intensity and LC retention time for quantitative analysis and precursor ion selection in proteomic LC-MALDI analyses.

This study describes a methodology for performing relative quantitation in large-scale proteomic sample comparisons using an LC-MALDI mass spectrometry analytical platform without the use of isotope tagging reagents. The method utilizes replicate analyses of a sample to create a profile of constituent components that are aligned based on LC elution time and mass. Once components from individual runs have been grouped as common "features", the Student's t test is used to determine which components are systematically different between samples. In this study, five HPLC runs of human plasma were compared to five HPLC runs of human serum. About 3889 components were detected in all 10 runs. Of these, 1831 corresponded to approximately 100 known serum proteins, based on MS/MS analysis of one run each from serum and plasma. As expected, fibrinogen alpha, beta, and gamma chains accounted for many of the most significant differences. Therefore, using MALDI, samples containing thousands of peptides can be compared in a minimal amount of time. Moreover, the results of the comparison can be used to guide further MS/MS mode sample interrogation in a result dependent manner.

Comparative study of [Three] LC-MALDI workflows for the analysis of complex proteomic samples.

Large-scale proteomic analyses frequently rely on high-resolution peptide separation of digested protein mixtures in multiple dimensions to achieve accuracy in sample detection and sensitivity in dynamic range of coverage. This study was undertaken to demonstrate the feasibility of MALDI MS/MS with off-line coupling to HPLC for the analysis of whole cell lysates of wild-type yeast by three different workflows: SCX-RPHPLC-MS/MS, high-pH SAX-RPHPLC-MS/MS and RP (protein)-SCX-RPHPLC-MS/MS. The purpose of these experiments was to demonstrate the effect of a workflow on the end results in terms of the number of proteins detected, the average peptide coverage of proteins, and the number of redundant peptide sequencing attempts. Using 60 microg of yeast lysate, minor differences were seen in the number of proteins detected by each method (800-1200). The most significant differences were observed in redundancy of MS/MS acquisitions.

Effect of solvent composition on signal intensity in liquid chromatography-matrix-assisted laser desorption ionization experiments.

The use of reversed phase liquid chromatography for the preparation of complex peptide mixtures for analysis by matrix assisted laser desorption ionization mass spectrometry has led to the observation of the critical importance of the matrix/analyte formulation in regards to the percent organic solvent in the mixture. This paper outlines the study using liquid chromatography to systematically vary the acetonitrile concentration in the formulation used for MALDI spot preparation to examine the impact the parameter has on analyte signal intensity. The results show that for five of six peptides tested across a wide mass range a formulation of approximately 75% acetonitrile is optimal for average MALDI signal intensity as determined on both time-of-flight and quadropole mass spectrometers. Examination of the individual spots shows that the organic solvent content in formulation significantly affects parameters such as crystal density and morphology.