Liquid Chromatography-Mass Spectrometry (LC-MS) for Biomarker Discovery

Biomarkers play an important role in drug development and help in the identification of disease-causing proteins found in blood, urine, body fluids, and tissue samples. The combination of liquid chromatography (LC) and mass spectrometry (MS) has been finding increasing clinical application in recent years in the area of biomarker discovery.

LC-MSImage Credit: Dermot McBrierty / Shutterstock.com

The high resolution of LC coupled with fast and sensitive MS methods has helped in the quantitative measurement of many proteins, isoforms, and post-translational modifications (PTM) in complex biological specimens, thereby cutting down on the huge expenditure that goes into developing specific immunoreagents.

Approaches to biomarker discovery through LC-MS

LC-MS is used for large-scale screening of potential protein biomarkers, known as the global proteomics method, as well as for targeted quantification of proteins, known as the targeted proteomics method. Quantification by the global proteomics method is carried out either by using stable isotopes for labeling or without labeling.

However, label-free protein quantification by LC-MS does not provide the required precision in the case of low-abundance peptides. The isotope labeling, in turn, provides precise quantification and does not depend on the reproducibility of LC-MS. Test samples with different labels can be co-eluted and analyzed by the LC-MS methods using isotope labels.

While both methods are used widely in the discovery of cancer biomarkers such as for ovarian, prostate, and renal carcinoma, targeted proteomics by LC-MS is quickly gaining recognition for its ability to quantify proteins accurately.

Recent researches

Neurodegenerative diseases and alcohol disorders:

LC-MS has seen application in the profiling of cerebrospinal fluid (CSF), with an accuracy of 83% and specificity of 100%, to identify protein pathways that are altered during neurodegenerative disorders such as amyotrophic lateral sclerosis.

A study by Isaksson et al. has demonstrated that LC-MS accurately detected phosphatidylethanol in whole blood (B-Peth) in alcohol disorders. B-Peth is an important biomarker to help diagnose alcohol disorders and for subsequent evaluation of treatment regimens.

Respiratory diseases:

Early and accurate diagnosis of reactive airway diseases is critical for effective treatment and management. Khami et al. have developed a high performance liquid chromatographic (HPLC)-MS method, validated by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), which is being tested for quantification of diagnostic biomarkers in urine for asthma and chronic obstructive pulmonary disease (COPD) using isotope labeling.

Human biomonitoring:

LC-MS has also been used to analyze consumer goods for the presence of hazardous phosphorus flame retardants and plasticizers (PFRs). Biomarkers such as triphenyl phosphate (TPHP), tris(2-chloroisopropyl) phosphate (TCIPP), tris(2-chloroethyl) phosphate (TCEP), and 2-ethylhexyldiphenyl phosphate (EHDPHP) were used to monitor the presence of PFRs in wastewater which could be helpful in human biomonitoring at a community level.

Biomarkers in the cell cycle:

Protein biomarkers have proven to be very valuable in the early detection of cell stress and death.

Using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS), Albrecht and his team translated and verified six proteins, namely glycerol-3-phosphate dehydrogenase (GPDH), galectin-1(LGALS1, peroxiredoxin 1 (PRDX1), transgelin-2 (TAGLN2), cofilin 1 (CFL1) and malate dehydrogenase (MDH), identified from a previous chinese hamster ovarian (CHO-K1) cell line.

This study confirmed the universality of markers in a cell growth model by growing CHO-S, CHO-K1, and CHO-DG44 cell lines in batch cultures using two types of basal media. By using stable isotope-labeled peptide analogs and monoclonal antibodies for enhanced protein quantification, a marked increase in protein concentrations was observed upon an increase in dead cell numbers.

This indicates the potential of using LC-MS in selecting viability marker proteins for understanding various biological processes.

Discovery of biomarkers for global health threats:

Carbapenemase-producing organisms (CPOs) carrying Klebsiella pneumonia carbapenemase (KPC) genes are continuing to be a global threat to public health. Scientists are now trying to develop rapid MS methods for detecting tryptic peptides in less than 90 minutes.

Wang et al. employed the genoproteomic method that combined theoretical analysis of peptidome with LC-MS to select three peptide markers of the KPC protein that can be detected accurately after rapid tryptic digestion. A blinded validation set comprising of KPC-positive and KPC-negative clinical isolates further validated the 100% sensitivity and specificity of this method.

Oncology:

LC-MS is widely being used in the field of oncology to predict treatment outcomes after chemotherapy, as well as for early detection of rare cancers such as esophageal adenocarcinoma. LC-MS has also been used to identify proteins that are secreted in the head, oral, and neck squamous cell carcinoma.

Ralhan and his team analyzed tumor cells for potential biomarkers by employing proteomic technologies and identified approximately 140 unique proteins. Analyzing the secretome of head and neck cancer cells can help in predicting the prognosis and subsequent evaluation of response to therapy by employing minimally invasive tests.

Biotherapeutics:

Of late, hybrid LC-MS has been used to analyze biomarkers and biotherapeutics wherein liquid binding immunoaffinity (LBA) is combined with LC-MS. LBA helps in enriching or selectively isolating the analyte of interest and has found application in many biotherapeutic areas such as messenger RNA therapeutics, carrier conjugates, antibody-drug conjugates, monoclonal antibodies, and so on.

Discovery of candidate peptides:

LC-MS has also been used by Longuespee R., et al. to help in reliable identification of proteolytic peptides that were obtained from matrix-assisted laser desorption/ionization (MALDI) imaging. The study also demonstrated a ten-fold reduction in the number of m/z species obtained by MALDI imaging by using LC-MS in combination. This proves the potential of LC-MS in discovering candidate peptides with further confirmation through literature review and immunohistochemical analysis.

Source:

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  • Sikorski T. Considerations for developing and validating an LC–MS biomarker assay using the surrogate peptide approach. Accessed on October 20, 2019. www.bioanalysis-zone.com/.../
  • Zhang J et al. (2012). Esophageal Cancer Metabolite Biomarkers Detected by LC-MS and NMR Methods. PLoS One. https://doi.org/10.1371/journal.pone.0030181
  • Collins MA, et al. (2015). Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis. J. Proteome Res. https://doi.org/10.1021/acs.jproteome.5b00804
  • Isaksoon A et al. (2017). High-Throughput LC-MS/MS Method for Determination of the Alcohol Use Biomarker Phosphatidylethanol in Clinical Samples by Use of a Simple Automated Extraction Procedure—Preanalytical and Analytical Conditions. American Association for Clinical Chemistry. DOI:10.1373/jalm.2017.024828
  • Khami MM et al. (2017). Development of a validated LC- MS/MS method for the quantification of 19 endogenous asthma/COPD potential urinary biomarkers. Analytica Chimica Acta. https://doi.org/10.1016/j.aca.2017.08.007
  • Been F et al. (2017. Liquid Chromatography–Tandem Mass Spectrometry Analysis of Biomarkers of Exposure to Phosphorus Flame Retardants in Wastewater to Monitor Community-Wide Exposure. Anal Chem. https://doi.org/10.1021/acs.analchem.7b02705
  • Albrecht S et al. (2018). Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control. Anal Bioanal Chem. DOI: 10.1007/s00216-018-1029-3.
  • Wang H et al. (2017). Peptide Markers for Rapid Detection of KPC Carbapenemase by LC-MS/MS. Scientific Reports. DOI:10.1038/s41598-017-02749-2
  • Ralhan R et al. (2011). Identification of proteins secreted by head and neck cancer cell lines using LC‐MS/MS: Strategy for discovery of candidate serological biomarkers. Proteomics. https://doi.org/10.1002/pmic.201000186
  • Yuan M et al. (2019). Hybrid Ligand Binding Immunoaffinity-Liquid Chromatography/Mass Spectrometry for Biotherapeutics and Biomarker Quantitation: How to develop a hybrid LBA-LC-MS/MS method for a protein? Beta science press. DOI: 10.17145/rss.19.005. (ISSN 2589-1677)
  • Longuespee R et al. (2018). Identification of MALDI Imaging Proteolytic Peptides Using LC‐MS/MS‐Based Biomarker Discovery Data: A Proof of Concept. Proteomics Clinical Applications. https://doi.org/10.1002/prca.201800158

Further Reading

Last Updated: Jan 8, 2020

Deepthi Sathyajith

Written by

Deepthi Sathyajith

Deepthi spent much of her early career working as a post-doctoral researcher in the field of pharmacognosy. She began her career in pharmacovigilance, where she worked on many global projects with some of the world's leading pharmaceutical companies. Deepthi is now a consultant scientific writer for a large pharmaceutical company and occasionally works with News-Medical, applying her expertise to a wide range of life sciences subjects.

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