Combined tissue and fluid proteomics with Tandem Mass Tags to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer's Disease case study.
BACKGROUND
Ideal biomarkers are present in readily accessible samples including plasma and cerebrospinal fluid (CSF), and are directly derived from diseased tissue, therefore likely to be of relatively low abundance. Traditional unbiased proteomic approaches for biomarker discovery have struggled to detect low-abundance markers due to the high dynamic range of proteins, the predominance of hyper-abundant proteins, and the use of data-dependent acquisition mass spectrometry (MS). To overcome these limitations and improve biomarker discovery in peripheral fluids, we have developed TMTcalibrator™; a novel MS workflow combining isobarically labelled diseased tissue digests in parallel with an appropriate set of labelled body fluids to increase the chance of identifying low-abundance, tissue-derived biomarkers.


METHODS
A disease relevant cell line was labelled with TMT® in a range of concentrations generating a multi-point calibration curve. Peripheral biofluid samples were labelled with the remaining tags and quantitative analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer with a Top10 CID-HCD MS3 synchronous precursor selection (SPS) method. SPS allowed direct analysis of non-depleted, unfractionated CSF samples with complete profiling of six individual samples requiring only 15 hours of MS time, equivalent to 1.5 h per sample.


RESULTS
Using the TMTcalibrator™ workflow allowed the identification of several markers of microglia activation that are differentially quantified in the CSF of patients with Alzheimer's disease (AD). We report peptides from 41 proteins that have not previously been detected in the CSF, that appear to be regulated by at least 60% in AD.


CONCLUSIONS
This study has demonstrated the benefits of the new TMTcalibrator™ workflow and the results suggest this is a suitable and efficient method of detecting low-abundance peptides within biological fluids. The use of TMTcalibrator™ in further biomarker discovery studies should be considered to overcome some of the limitations commonly associated with more conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.
Proteome Sciences plc, Institute of Psychiatry, Kings College London, South Wing Laboratories, De Crespigny Park, London, SE5 8AF, UK.
Claire L
CL
Russell
Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.
Amanda
A
Heslegrave
Proteome Sciences plc, Institute of Psychiatry, Kings College London, South Wing Laboratories, De Crespigny Park, London, SE5 8AF, UK.
Vikram
V
Mitra
Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.|Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden.
Henrik
H
Zetterberg
Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden.
Jennifer M
JM
Pocock
Proteome Sciences plc, Institute of Psychiatry, Kings College London, South Wing Laboratories, De Crespigny Park, London, SE5 8AF, UK.
Malcolm A
MA
Ward
Proteome Sciences plc, Institute of Psychiatry, Kings College London, South Wing Laboratories, De Crespigny Park, London, SE5 8AF, UK.
Ian
I
Pike
D000544:Alzheimer Disease / Q000134:cerebrospinal fluid*; D000818:Animals; D015415:Biomarkers / Q000134:cerebrospinal fluid*; D002460:Cell Line; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000134:cerebrospinal fluid; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015415:Biomarkers; D010446:Peptide Fragments; D020543:Proteome