Name: sample preparation for endopeptidomic analysis in human cerebrospinal fluid
Uploaded: 2017-12-04T13:00:00
Description: Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

Many thanks to Tanveer Batth and colleagues for advice in setting up the pre-fractionation method.
This work was supported by funding from the Swedish Research Council, the Wallstr&#246;m and Sj&#246;blom Foundation, the Gun and Bertil Stohne Foundation Stiftelse, the Magnus Bergwall Foundation, the &#197;hl&#233;n Foundation, Alzheimerfonden, Demensf&#246;rbundet, Stiftelsen f&#246;r Gamla Tj&#228;narinnor, the Knut and Alice Wallenberg Foundation, Frimurarestiftelsen, and FoU-V&#228;stra G&#246;talandsregionen.
The main recipients of funding for this project were Kaj Blennow, Henrik Zetterberg and Johan Gobom.

&#160;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 materia...

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonality+of+separation+in+two-dimensional+liquid+chromatography.">Orthogonality of separation in two-dimensional liquid chromatography.</a> Anal Chem. 77 (19), 6426-6434 (2005).</li><li>Batth, T. S., Francavilla, C., Olsen, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-line+high-pH+reversed-phase+fractionation+for+in-depth+phosphoproteomics.">Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.</a> J Proteome Res. 13 (12), 6176-6186 (2014).</li><li>Yang, F., Shen, Y., Camp, D. G., Smith, R. 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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...

Biomarkers in cerebrospinal fluid (CSF) are currently transforming research into neurodegenerative disorders. In Alzheimer&#39;s disease, the most common neurodegenerative disorder, affecting over 60 million people worldwide1,2, 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&#39;s disease and Multiple Sclerosis, proteomic studies have identified numerous biomarker candidates, some of which are currently under evaluation in clinical studies4,5,6.
Alongside proteins, CSF also contains an abundance of endogenous peptides7,8,9,10,11,12. Constituting cleavage products of many brain-derived proteins, these peptides also represent a potentially important source of disease biomarkers. To increase the inventory of identified endogenous peptides in human CSF and enable CSF endopeptidomic analyses in clinical studies, a method was developed for sample preparation and LC-MS analysis (a brief protocol scheme has been included in Figure 1).The application of this method in a recent study resulted in the identification of nearly 16,400 endogenous CSF peptides in pooled CSF samples from several individuals of non-specific diagnosis, expanding the known CSF endopeptidome ten-fold13. The method can optionally be used in conjunction with isobaric labelling approach for quantification.
Sample Preparation
The main source of protein mass in CSF is plasma constituents (e.g. albumin and immunoglobulins) passing over the blood brain barrier14,15. Their high abundance hampers the detection of low-abundant, brain-derived sample components. Endogenous peptides can be readily separated from the high-abundant proteins, thereby allowing a significantly larger volume of CSF peptide extract to be used for LC-MS analysis, thereby enabling detection of lower-abundant peptides.
In the protocol presented here, molecular weight cut-off (MWCO) filtration was used to separate the CSF peptides from the protein fraction; a method that has been used in several previous studies8,9,10,11,12,16. The filtration step was followed by an offline RP HPLC pre-fractionation step performed over a high-pH mobile phase gradient. By performing two RP HPLC steps in tandem, with pH being the main distinction, the difference in selectivity between the two steps results mainly from altered peptide retention as a consequence of different peptide charge states. The application of high-pH peptide pre-fractionation prior to LC-MS under acidic conditions has proven efficient in increasing peptide identification17,18, and even to be superior for this purpose in complex biological samples compared to more orthogonal separation modes19, such as strong cat-ion exchange (SCX) and RP20. To shorten the analysis time, a concatenation scheme was used, pooling every 12th fraction (e.g., fractions 1, 13, 25, 37, and 49), which due to the high resolving power of RP HPLC still largely avoided co-elution of peptides from different fractions in the LC-MS step20,21.
Peptide identification
Peptide identification in peptidomic studies differs from that of proteomic studies in that no enzyme cleavage can be specified in the database search, and as a consequence, identification rates are usually lower11. A recent study13&#160;showed that the identification rates for endogenous peptides obtained with Sequest and Mascot were substantially improved when the default scoring algorithm of the respective software program was modified using the adaptive scoring algorithm Percolator, indicating that optimal scoring algorithms for endogenous peptides differ from that of tryptic peptides13. In that study, identification based on automatic peptide de novo sequencing using the software PEAKS (BSI) was found to be complementary to the two fragment ion fingerprinting-based search engines, resulting in a significantly larger set of identified peptides.

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		<tr height="38">
			<td height="38" style="height:38px;width:547px;">&#160;1 M Triethylammonium bicarbonate</td>
			<td style="width:339px;">Fluka, Sigma-Aldrich</td>
			<td style="width:339px;">17902-100ML</td>
			<td style="width:339px;">TEAB</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">&#160;8 M Guanidinium hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G7294-100ML</td>
			<td>GdnHCl</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Pierce</td>
			<td>20490</td>
			<td>TCEP</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td>I1149-5G</td>
			<td>IAA</td>
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		<tr height="38">
			<td height="38" style="height:38px;">Hydroxylamine 50% (w/w)</td>
			<td>Sigma-Aldrich</td>
			<td>457804-50ML</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acetonitrile, Far UV, HPLC gradient grade</td>
			<td>Sigma-Aldrich</td>
			<td>271004-2L</td>
			<td>AcN</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Formic acid</td>
			<td>Fluka, Sigma-Aldrich&#160;</td>
			<td>56302-1mL-F</td>
			<td>FA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Triflouroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508-10AMP</td>
			<td>TFA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>30501-1L-1M</td>
			<td>NH4OH</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane</td>
			<td>Merck Millipore</td>
			<td>UFC903024</td>
			<td>MWCO-filter</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sep-Pak C18, 100 mg</td>
			<td>Waters</td>
			<td>WAT023590</td>
			<td>SPE-column</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Resprep 12-port SPE Manifold&#160;</td>
			<td>Restek</td>
			<td>26077</td>
			<td>Vacuum manifold</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">TMT10plex Isobaric Label Reagent Set</td>
			<td>Thermo Fisher Scientific</td>
			<td>90110</td>
			<td>TMT10plex</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">UltiMate 3000 RSLCnano LC System</td>
			<td>Dionex</td>
			<td>5200.0356</td>
			<td>Online sample separation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ultimate 3000 RPLC Rapid Separation Binary System</td>
			<td>Dionex</td>
			<td>IQLAAAGABHFAPBMBEZ</td>
			<td>Offline high-pH fractionation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Orbitrap Fusion Tribrid mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td style="width:339px;">IQLAAEGAAPFADBMBCX</td>
			<td>Mass spectrometer for sample analysis</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Proteome Discoverer 2.0&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGABSFAKJMAUH</td>
			<td>Proteomics search platform</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Mascot v2.4</td>
			<td>Matrix Science</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sequest HT</td>
			<td>Thermo</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">PEAKS v7.5&#160;</td>
			<td>Bioinformatic Solutions Inc.)</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap 100, 75 &#181;m x 2 cm, C18, 100 &#197; pore size, 3 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164535</td>
			<td>Trap column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap C18, 75 &#181;m x 500 mm, 100&#197; pore size,&#160;2 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164942</td>
			<td>Separation Column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Savant SpeedVac High Capacity Concentrators</td>
			<td>Thermo Fisher Scientific</td>
			<td>SC210A-230</td>
			<td>SpeedVac/Vacuum concentrator</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">XBridge Peptide BEH C18 Column, 130&#197;, 3.5 &#181;m, 2.1 mm X 250 mm</td>
			<td>Waters</td>
			<td>186003566</td>
			<td>Separation Column (micro HPLC)</td>
		</tr>
	</tbody>
</table>

sample preparation for endopeptidomic analysis in human cerebrospinal fluid

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.

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<sup class="x...

Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

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.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

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A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of AÃƒÅ½Ã‚Â² and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...