Developing an iMALDI method.

The iMALDI (immuno-MALDI) technique involves the affinity capture of target peptides from an enzymatic digest of a sample, followed by the direct analysis of the affinity beads while on a MALDI target. For determination of peptide concentration (and, by inference, protein concentration), stable-isotope-labeled standard peptides (SIS peptides) can be added to the digest and will be captured along with the native peptides. This technique can provide the highest possible specificity by determining two molecular characteristics of the epitope-containing peptides: (1) the molecular weight, typically measured to within 100 ppm or better by MALDI-MS, and (2) the amino acid sequence, by performing MALDI-MS/MS. This technique has been shown to be capable of detecting low-attomole levels of target peptides in environmental samples and in digests of human plasma. This chapter provides a detailed description of how to perform iMALDI analyses, starting with the selection of the target peptides. Examples are shown of the application of iMALDI to the detection of an organism that is a possible bioterrorism threat, and to the detection of two isoforms of human EGFR.

Duplexed iMALDI for the detection of angiotensin I and angiotensin II.

An immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) method has been developed using anti-IgG beads to capture anti-AngI and anti-AngII antibodies, which are incubated with a ∼50µL plasma sample to which known amounts of stable-isotope-labeled AngI and AngII have been added. After a short incubation time, the beads are washed, placed directly on a MALDI target, and analyzed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The iMALDI assay developed can detect and quantify angiotensin I (AngI) and angiotensin II (AngII) in human plasma. This assay has a Limit of Detection (LOD) of ∼10amol/µL (or ∼13pg/mL AngI and ∼11pg/mL AngII), at a S/N of 2:1, using only one-tenth of the antibody beads which were incubated with a 50-µL plasma sample. This LOD is within the relevant range of patient samples. Little or no angiotensin generation period is required, resulting in a rapid assay. Correlation coefficients for the standard curves are >0.99, with a linear range of 4-100fmol/µL (5-130ng/mL) and 100-2500amol/µL (106-2614pg/mL) for AngI and AngII, respectively. This duplexed assay can quantify AngI and AngII peptide levels simultaneously, in plasma from normotensive and hypertensive patients. The assay can detect changes in the levels of these peptides over time, which will allow quantitation of plasma renin and ACE activities.

Towards the development of an immuno MALDI (iMALDI) mass spectrometry assay for the diagnosis of hypertension.

The renin-angiotensin-aldosterone system (RAAS) plays an essential role in the regulation of plasma volume and arterial blood pressure. One of the most common diseases of the RAAS is the autonomous production of aldosterone by the adrenal glands, caused by either bilateral adrenal hyperplasia or an aldosterone-producing adenoma. This condition, known as primary aldosteronism, is a treatable and often curable form of hypertension. The measurement of plasma renin activity (PRA), as determined by radioimmunoassay for angiotensin I is essential to the diagnosis of primary aldosteronism. However, accurate determination of PRA is often hampered by low plasma concentrations of angiotensin I. Here, we report the use of immuno-MALDI (iMALDI) as a highly sensitive and specific method for the absolute quantitation of angiotensin I in plasma. iMALDI permits concentration determination by affinity-capture of angiotensin I and a stable-isotopically labeled standard (SIS) peptide on immobilized anti-peptide antibodies. The affinity beads are placed on the MALDI target, permitting automated analysis of large numbers of patient samples. Pretreatment of the plasma is not required, and this method is suitable for the accurate determination of angiotensin I in whole plasma. The calibration curve generated using this method was linear over a 50-fold concentration range in plasma, with a correlation coefficient of 0.984. MS/MS sequence confirmation provides absolute specificity. The iMALDI angiotensin I assay, therefore, has the potential to be developed into a method for determining PRA that has advantages in time, in specificity, and in safety.

Protein arrays for biomarker discovery.

The value of biomarker research in the field of diagnostics and predictive medicine has long been acknowledged. Diagnostic tests have evolved from single-molecule monitoring to multiplexed assays for entire panels of biomarkers, resulting in comprehensive data that clinicians can use to efficiently and effectively treat patients. Thus, the focus of protein microarrays has been directed toward the development of high-throughput multiplex assays that are capable of producing highly accurate quantitative data for a dynamic range of molecules to meet the requirements of the clinical laboratory. Some recent advancements in the field of biomarker discovery, including mass-spectrometry based approaches, as well as the advantages and limitations of the various techniques, will be discussed in this review.