Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

Summary

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

Abstract

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.

Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.

Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.

Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

Introduction

Biomarkers in cerebrospinal fluid (CSF) are currently transforming research into neurodegenerative disorders. In Alzheimer's disease, the most common neurodegenerative disorder, affecting over 60 million people worldwide^1,2, a biomarker triplet consisting of the peptide amyloid beta, microtubule-stabilizing protein tau, and a phosphorylated tau form, can detect the disease with high sensitivity and specificity, and has been included in the diagnostic research criteria³. In other neurodegenerative diseases, such as Parkinson's disease and Multiple Sclerosis, proteomic studies hav....

Protocol

 The protocol described below is a refined version of the one used in a previous study where a large amount of endogenous peptides were identified in human CSF¹⁵. Updates to the original protocol involve minor alterations to the chemical pre-treatment of CSF as well as optimisation of the gradient used for offline high-pH RP HPLC pre-fractionation.

Ethical considerations

All studies of Swedish patient and control materials have been approved by ethical committees: St. Göran (ref. 2005-554-31/3). CSF samples from the Amsterdam Dementia Cohort and samples collected....

Results

The method described here has been applied and evaluated in three studies prior to the introduction of sample pre-fractionation (Table 1). The first study used offline LC for spotting CSF fractions on a MALDI target plate and resulted in 730 identified endogenous peptides¹¹. In the two following studies, isobaric labelling was employed. Primarily in a case/control study for identification and characterisation of potential biomarkers in the CSF endopeptidome and proteome simultaneously

Discussion

The introduction of an high-pH RP HPLC pre-fractionation step to a previously developed protocol for recovery of endogenous peptides by molecular weight ultrafiltration¹¹ reduced relative sample complexity and thereby allowed for a 5-fold larger sample volume to be studied. This, in turn, increased the concentration of the subset of peptides present in each fraction and thereby improved the chances of detecting low abundant peptides.

By performing an identification stra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 M Triethylammonium bicarbonate	Fluka, Sigma-Aldrich	17902-100ML	TEAB
8 M Guanidinium hydrochloride	Sigma-Aldrich	G7294-100ML	GdnHCl
Tris(2-carboxyethyl)phosphine hydrochloride	Pierce	20490	TCEP
Iodoacetamide	SIGMA	I1149-5G	IAA
Hydroxylamine 50% (w/w)	Sigma-Aldrich	457804-50ML
Acetonitrile, Far UV, HPLC gradient grade	Sigma-Aldrich	271004-2L	AcN
Formic acid	Fluka, Sigma-Aldrich	56302-1mL-F	FA
Triflouroacetic acid	Sigma-Aldrich	T6508-10AMP	TFA
Ammonium hydroxide solution	Sigma-Aldrich	30501-1L-1M	NH4OH
Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane	Merck Millipore	UFC903024	MWCO-filter
Sep-Pak C18, 100 mg	Waters	WAT023590	SPE-column
Resprep 12-port SPE Manifold	Restek	26077	Vacuum manifold
TMT10plex Isobaric Label Reagent Set	Thermo Fisher Scientific	90110	TMT10plex
UltiMate 3000 RSLCnano LC System	Dionex	5200.0356	Online sample separation
Ultimate 3000 RPLC Rapid Separation Binary System	Dionex	IQLAAAGABHFAPBMBEZ	Offline high-pH fractionation
Orbitrap Fusion Tribrid mass spectrometer	Thermo Scientific	IQLAAEGAAPFADBMBCX	Mass spectrometer for sample analysis
Proteome Discoverer 2.0	Thermo Fisher Scientific	IQLAAEGABSFAKJMAUH	Proteomics search platform
Mascot v2.4	Matrix Science	-	Proteomics search engine
Sequest HT	Thermo	-	Proteomics search engine
PEAKS v7.5	Bioinformatic Solutions Inc.)	-	Proteomics search engine
Acclaim PepMap 100, 75 µm x 2 cm, C18, 100 Å pore size, 3 µm particle size	Thermo Fisher Scientific	164535	Trap column (nano HPLC)
Acclaim PepMap C18, 75 µm x 500 mm, 100Å pore size, 2 µm particle size	Thermo Fisher Scientific	164942	Separation Column (nano HPLC)
Savant SpeedVac High Capacity Concentrators	Thermo Fisher Scientific	SC210A-230	SpeedVac/Vacuum concentrator
XBridge Peptide BEH C18 Column, 130Å, 3.5 µm, 2.1 mm X 250 mm	Waters	186003566	Separation Column (micro HPLC)