Application of mass spectrometry to molecular diagnostics of viral infections

Abstract

Mass spectrometry (MS) has found numerous applications in life sciences. It has high accuracy, sensitivity and wide dynamic range in addition to medium- to high-throughput capabilities. These features make MS a superior platform for analysis of various biomolecules including proteins, lipids, nucleic acids and carbohydrates. Until recently, MS was applied for protein detection and characterization. During the last decade, however, MS has successfully been used for molecular diagnostics of microbial and viral infections with the most notable applications being identification of pathogens, genomic sequencing, mutation detection, DNA methylation analysis, tracking of transmissions, and characterization of genetic heterogeneity. These new developments vastly expand the MS application from experimental research to public health and clinical fields. Matching of molecular techniques with specific requirements of the major MS platforms has produced powerful technologies for molecular diagnostics, which will further benefit from coupling with computational tools for extracting clinical information from MS-derived data.

Figure 1. Basic configuration of an ESI mass spectrometer for use in nucleic acid analysis

LC: Liquid chromatography; MS: Mass spectrometry.

Figure 2. Basic configuration of a MALDI mass spectrometer for use in nucleic acid analysis

MS: Mass spectrometry.

Figure 3. Biochemical processes for comprehensive molecular analysis of specific gene targets as applied to analysis by mass spectrometry

(A) Restriction fragment length polymorphism – the complexity of large target molecules is reduced by enzymatic cleavage at predetermined sites, the resulting small molecules are analyzed by MS and the obtained mass patterns are used to infer organism identification. (B) Mass-tag multiplexing – each target is detected by a probe tagged by a cleavable small molecule, unused probes are removed/washed, the tags of the used probes are cleaved and used for detection by MS; detected tags indicate presence of the target of interest. (C) Generation of sequence ladder in the presence of one specific primer, regular nucleotides, dideoxynucleotides and polymerase, resulting in random termination of the fragments at any and all positions, allowing the discrimination of two fragments by one added nucleotide. (D) Sequencing by transcription with T6 and SP6 polymerases, coupled to RNase A single base-specific cleavage. The process queries both strands of each amplicon of interest. The resulting mass fingerprint contains information about all four nucleotides and is used to derive the fragment’s sequence, genotype and heterogeneity and to discover new mutations by in silico pattern comparison to a comprehensive reference set. (E) SNP identification by multiplex PCR. Each product is then queried by a specific probe designed immediately upstream from a SNP of interest and then extended in the presence of ddNTP mix. The resulting extended probes present a mass signature that identifies the SNP sequence. Depending on the assay design (e.g., T5000) the multiplex product could be used directly for MS with the resulting mass patterns used for organism identification. MS: Mass spectrometry; SAP: Shrimp alkaline phosphatase.

Figure 4. Process pipeline for multiplex pathogen detection and identification by nucleic acid testing on an ESI platform

(A) Processes that are performed off the MS platform. (B) Processes that are executed on the MS platform. MS: Mass spectrometry; NA: Nucleic acid.

Figure 5. Process pipeline for multiplex single nucleotide polymorphism detection/identification and nucleic acid sequencing by testing on a MALDI-TOF platform

The top panel of A and B processes are performed off the MS platform, and then followed by acquisition executed on the MS platform. (A) Process line provides multiplexed SNP detection and identification. (B) Process line provides sequencing information. The estimated samples/hour refers to the capacity of the MS. MS: Mass spectrometry; NA: Nucleic acid.