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Precision Nanomedicine

The official journal of CLINAM

About the Journal: Precision Nanomedicine (PRNANO) is a peer-reviewed, not-for-profit, international society journal to promote all practical, rational, and progressive aspects of theory and practice of nanomedicine, from basic research through translational and clinical aspects including commercialization.

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A coming era of precision diagnostics based on nano-assisted mass spectrometry

Rongxin Li1†, Deepanjali Dattatray Gurav2†, Jingjing Wan1*, and Kun Qian2[1]*

1School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, 200062, P. R. China

2School of Biomedical Engineering, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, 200030, P. R. China

These authors contributed equally to this work.

Submitted: July 24, 2018; Accepted: July 31, 2018

Keywords

Diagnostics, mass spectrometry, nanotechnology, omics

Abbreviations:

  • SNP single-nucleotide polymorphism
  • qPCR quantitative real-time polymerase chain reaction
  • a6A N6-allyladenosine
  • RT reverse transcriptase
  • CyTOF mass cytometry by time-of-flight
  • Pre-DC dendritic cells precursors

License:

CC BY-NC-SA 4.0

Abstract

Precision diagnostics relies on omic analysis by mass spectrometry to overcome the limitation in accuracy by an individual biomarker, due to the complex nature of diseases. Recent development in nanotechnology markedly enhanced sample treatment and detection efficiency of this method. Herein, we foresee a coming era of precision diagnostics based on nano-assisted mass spectrometry. Some important progress in the field includes detection of (1) nucleic acids for genetic analysis; (2) proteins/peptides for proteomic analysis; and (3) small molecules for metabolic analysis. We anticipate that this review will be a reminder for both young and experienced researchers about the future of diagnostics and call for attention worldwide.

Purpose and Rationale

Diagnostics is the core of biomedical research and clinical practice, which guides the prevention and treatment of diseases for better healthcare globally.1-3 Notably, precision diagnostics relies on omic analysis to overcome the limitation in accuracy by an individual biomarker,4-7 due to the complex nature of physiological and pathological process.1,8,9 Among numerous analytical approaches for omics, mass spectrometry (MS) has become the major tool due to the desirable throughput, sensitivity, and identification capability.10-13 To date, the state-of-art MS methods and techniques have advanced omics in all levels including genomics,14,15 proteomics,16-18 and metabolomics.19-21

Nowadays, interdisciplinary research is fundamental to achieve real case applications of MS in clinics. Particularly, development of nanotechnology enhanced sample treatment and detection efficiency of MS for diagnostics.10,22-24 For sample treatment, nanoscale materials and devices afford unique size-dependent properties for selective extraction of either a specific molecule or a group of molecules.3,20,25,26 For detection efficiency, nanoscale materials and devices contribute to the amplified ionization performance with orders of magnitude.24,25,27 Therefore, nanotechnology can tackle the key obstacles for MS-based omics, which can be further combined with instrumentation and data mining for next generation of precision diagnostics.

Herein, we foresee a coming era of precision diagnostics based on nano-assisted MS. We show a few important developments in the field (Scheme 1 and Table 1), including MS detection of (1) nucleic acids for genetic analysis; (2) proteins/peptides for proteomic analysis; and (3) small molecules for metabolic analysis. We anticipated that this review would be a reminder for both young and experienced researcher about the future of diagnostics, calling for the attentions worldwide.

Scheme1

Scheme 1. Schematic illustration of omic analysis including genomics, proteomics, and metabolomics based on nano-assisted mass spectrometry.

Table 1 Overview of nano-assisted MS and typical applications that could be replaced

Categories for nano-assisted MS

Performance of nano-assisted MS

Typical applications that could be replaced

Performance of typical applications

Nano-MS based genomics

sensitivity: high

throughput: high

cost: moderate

quantitation: yes

gene chip[31]

quantitative real-time PCR[33]

sanger gene sequencing[34]

sensitivity: high

throughput: low

cost: very high

quantitation: yes

Nano-MS based proteomics

sensitivity: high

throughput: high

cost: moderate

quantitation: yes

chromatography[38]

protein microarrays[39]

gel electrophoresis[40]

sensitivity: high

throughput: moderate

cost: high

quantitation: yes

Nano-MS based metabolomics

sensitivity: very high

throughput: very high

cost: low

quantitation: yes

biochemical analyzer[47]

NMR[48]

electrochemical sensing[49]

sensitivity: high

throughput: low

cost: moderate

quantitation: yes

MS = mass spectrometry; PCR = polymerase chain reaction.

Summary of relevant literature and discussion

Detection of nucleic acids for genetic analysis

Genomics is the study of nucleic acids for genetic evaluation of an organism, analyzing the structure and function of genomes.28,29 Compared to the well-defined analysis of mutations at DNA level, the emerging fields of RNA editing, methylation, and splicing generate a library of transcript isoforms encoding genetic information from DNA-RNA-proteins.30 RNA editing is a molecular process effectively altering the amino acid sequence of the encoded protein to produce a new genomic sequence. It is crucial to develop “omics approach” for RNA editing, since RNA editing deals with modifying the nucleotide sequence in a specific genomic template to produce a new nucleotide sequence for understanding the molecular mechanisms of disease such as cancer. Deciphering these mutated sequences contributes to the development of disease-specific prognostic and therapeutic approaches. Different analytical methods have been employed for detailed analysis of genetic sequences such as gene chips,31 single-nucleotide polymorphism (SNP) microarrays,32 quantitative real-time polymerase chain reaction (qPCR),33 sanger gene sequencing,34 and so on. Among these, SNP microarray genotyping is a tool to determine genetic mutations, phenotype-specific panels, and genome-wide panels of a particular individual.32

1

Figure 1. Mass spectrometry detection of nucleic acids for genetic analysis. Schematic illustration of Nm-seq method based on oxidative cleavage for mapping 2′-O-methylation with base precision. (i) was sequencing process and (ii) showed the obtained mass spectra. Reproduced with permission.[14] Copyright 2017, Springer Nature.

Combining MS with these microarray techniques provides the ability for rapid, accurate, and quantitative characterization of post-transcriptionally modified nucleosides based on their molecular weight changes. Hence, the continued improvements in the methodology and instrumentation used for the mass spectral analysis of nucleic acids will increase the applications of this technology to the field of genomics.

MS-based microarrays enable hybridization free analysis of nucleic acids based on their respective molecular weights. Research groups lead by Chuan He and Jianzhao Liu reported a new small molecule called as N6-allyladenosine (a6A) for RNA (ribonucleic acid) labeling through both metabolic and enzyme-assisted manners (Figure 1).15

The total extracted RNAs were digested into single nucleosides and analyzed by ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometer (UHPLC-QQQ-MS/MS) for accurate quantification of a6A levels. Notably, the increased molar mass of a modified probe by MS quantification validated the proposed RNA post-treatment reaction mechanism. Overall, the metabolically incorporated a6A molecule provides facile differentiation of labeled and unlabeled RNA using reverse transcriptase (RT)-induced mutation assay for its potential sequencing applications in the RNA field. Research work by Dai et. al. utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) for accurate detection and quantification of 2′-O-methylated (Nm) sites in mRNA molecules at low stoichiometry (Figure 2).14

2

Figure 2. RNA labeling study for genetic analysis. (i) Test of the allyl transfer ability. (ii) Mass spectra of modified RNA oligo and percentages of mutation. Adapted with permission[15] Copyright 2017, American Chemical Society.

The MALDI-TOF spectra of modified and unmodified standard RNA oligonucleotides revealed the feasibility of RNA modification. A conceptually distinct approach was developed based on the different chemical properties of nucleosides with 2′-OH and 2′-OMe groups by periodate oxidation for exposing, enriching and mapping Nm sites in the transcriptome with single-nucleotide precision. Thus, deciphering the deregulated RNA processing events facilitates the detection of rare but functionally relevant transcripts at cellular level. The RNA editing studies will provide key insights for detecting disease-specific biomarkers and development of novel therapeutic strategies.

Detection of proteins/peptides for proteomic analysis

Proteomics focuses on the large-scale study of proteins produced or modified at the gene or cellular level by an organism. Proteomics is more complicated owing to the distinct gene expressions in every system.6,35,36 Proteins have been detected using variety of techniques such as antibody free/ labeled immunoassays,37 chromatographic techniques,38 or protein microarrays,39 gel electrophoresis,40 and mass spectrometric methods prominently Orbitrap, matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI).3,16,41 MS-based proteomics is an advanced technology interpreting encoded information in the genomes.35,42,43 The approach has been successful in the case of small sets of proteins isolated in specific functional contexts.44 Detecting patterns of a differentially expressed proteins clinical samples shows the potential to diagnose the presence and stage of many diseases such as cancer.35,45 Thus, the ability of MS to precisely identify and quantify thousands of proteins from complex samples has broadly impacted biology and medicine.

Hu and co-workers have developed a method using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) for detection of specific peptide fragments present in Mycobacterium tuberculosis (Mtb) using a high-throughput MS approach (Figure 3).16