SAMPLE PREPARATION; The Achilles Heel of Mass Spectrometry Based Diagnostics

Regnier, Fred

Abstract

There is great excitement today about the use of mass spectrometry (MS) in clinical diagnostics, as we know from therapeutic drug monitoring. With ?L volumes of plasma, modern MS can identify and quantify a hundred different drugs, metabolites, and proteins in seconds at sensitivities approaching ELISA. But MS goes far beyond ELISA assays. With coupled immunosorbent capture, MS can differentiate between proteoforms of a protein in seconds; identifying disease associated isoforms from among perhaps 50 very similar structures. ELISA can't do that. Single amino acid polymorphism is similar; in Factor XIII a Trp187Arg substitution leads to a hemorrhagic disorder in neonates that is easily recognized by MS. Clearly, MS analysis of ?L blood volumes by immune selection (or some other type of structure specific affinity selection) will become the norm in diagnostics. Unfortunately blood is so complex it overwhelms MS instruments. Samples must be prepared in a laboratory by centrifugation and multiple preparation steps before MS analysis. More than 500 million blood samples are analyzed annually in the US using old sample preparation methods. The goal of this proposal is to miniaturize, simplify, and accelerate this process by developing a new sample preparation system that 1) extracts plasma from a finger-stick derived drop of blood, 2) collects 3.5 ?L plasma aliquots, 3) adds labeled internal standards, 4) affinity capture analytes with nanosorbents in a collection membrane, 5) chemically modifies analytes in some cases, 6) purifies analytes in a 60 sec time frame, and 7) introduces samples into an MS. Technological leaps such as this will change clinical diagnostics; giving subjects at remote sites access to sophisticated MS analyses of their blood through transport of a dried sample collection disc the size of a postage stamp to analytical laboratory. Last year Novilytic introduced the Noviplex microfluidic sample preparation card consisting of a membrane stack that extracts 2.5 ?L of plasma by capillary action from a finger-stick derived drop of blood. Preliminary studies with new, more advanced versions of Noviplex have shown that steps 1-5 can be executed automatically within a single small card without an energy source; enabling sampling at remote sites via a finger-stick, a substantial amount of sample preparation within each card, and dry transport to the analytical laboratory in a disc smaller than a postage stamp. With a large portion of the world's population far from advanced diagnostic laboratories, ease of sample transport is a major issue. We are proposing to develop technology that allows the first stages of sample preparation within Noviplex cards in-transit and final preparation in the LC-MS/MS platform; in-transit meaning post-plasma extraction An enabling feature is a new form of chromatography we will develop referred to here as mobile-sorbent affinity chromatography (MSAC) which involves the use of nanoscale affinity chromatography sorbents (NACS) in the mobile phase of chromatography columns. These nanosorbents will be synthesized in sizes too large to enter 50 nm pores in chromatography sorbents. NACS will be dispersed in water and added to the collection membrane on Noviplex cards where they will strongly sequester analytes within minutes. When the card arrives in the analytical lab, NACS will be eluted from the card and separated from other sample components in the LC-MS/MS by a C-18 restricted access media (RAM) column packed with particles of <50 nm pore diameter. NACS will be transported through the RAM column unretained at the velocity of the mobile phase while unbound substances are stripped from samples by the C-18 phase. Analytes will be recovered from NACS by denaturation of the affinity agent within the LC flow-train and eluted through a second RAM column where they are adsorbed and enriched while sorbents are transported to waste. Identification and quantification will be achieved by ballistic gradient elution of the secod RAM column into an MS in <60 sec. This allows the equivalent of many ELISA tests every min; an achievement of great significance in diagnostics.

Public Health Relevance

Even though the advance of mass spectrometry allows us to analyze small molecules or peptides in seconds, sample preparation becomes a rate-limiting step for the Metabolomics and Proteomics. The nanoparticulate affinity chromatography sorbents (NPACS) will be developed to purify target analytes from the plasma, which is combined with a restricted access media (RAM) column to isolate the selected analytes while they are transported in a mobile phase. The goal of the research is to develop the new system for mass linked immune-selective analysis (MALISA), where the eluted analyte can be identified and quantified by a mass spectrometer (MS) in minutes. This technology implements a new paradigm where sample preparations are directly connected to high throughput mass spectrometry.