cumulomics optimization of sample preparation for low amount … · 2014. 12. 11. · descriptive...

Cumulomics ‐ Optimization of sample preparation for low amount cumulus samplesClaudia Fortes1; Bernd Roschitzki1; Jasmin Walter2; Ralph Schlapbach1

1FGCZ, University Zurich/ETH Zurich, Zurich, Switzerland; 2Vetsuisse Faculty, Zurich, Switzerland

IntroductionLow success rates in in‐vitro fertilizationof livestock leads to high interest fromclinical side i.e. veterinary medicine toinvestigate the interaction between thecumulus cells and their oocyte in acumulus oocyte complex. Shotgunproteomics can provide an insight tothe protein networks involved infertilization. The big challenge of suchan investigation is the highly reducedamount of sample to be processed andanalyzed. The focus of this study is toelucidate sample handling protocols forshotgun proteomics of very limitingsample amount.

Overview• Evaluation of a set of methods which

are assumed to be appropriate forlow sample amount handling.

• Identification of the most suitablemethod by performing somestatistics on the outcome, such ascleavage performance, sensitivityfor Modifications and supposedprotein bias.

• Sample preparation of a clinical lowamount cumulus sample with thebest performing method evaluatedby the preliminary tests.

MethodsSample preparation was optimized using 2ug yeast extract for each preparation. Samples were prepared as shown in Fig 1. Eachpeptide extract was cleaned on C18 SPE. Peptides were analyzed on a Obritrap Q‐Exactive, coupled to Easy‐LC (Gradient:120min, 1% ‐ 35% acetonitrile in 0.1% formic acid, Column material: RP C18 AQ , 1.9um, 150x0.75mm, 50Celsius). Data analysiswas performed using Mascot 2.4.1 and ProteinPilot ™ 4.5. Sample preparation performance was analyzed using ProteinPilot™Descriptive Statistics Template (PDST).Bovine cumulus samples were obtained from single oocytes. Cells from individual oocytes were counted in Neubauer chambergiving input material amounts of 260 cells in the compact and 215 cells in the expanded cumulus samples. The samples weresubjected to SR‐FASP. Data acquisition was performed as written above. For fold change analysis in compact and expandedcumulus we have used Scaffold™ (95% protein prob. 2 peptides, Mascot ion score >50).

Figure 1. Generalized Method Overview.  

FASPSRADIn Solution SR‐FASP

95°C95°C

DTT Urea; TCEP SDS; Urea SDS; Urea

IAA

DTT

ClAA

DTT

Trypsin; RapigestIAA; Urea

Urea

Trypsin; Rapigest

Urea

IAA; Urea

Trypsin Trypsin

Figure 2. PDST evaluation of the preliminary tests(A) SRAD[3] could not reach the same performance as a regular

In‐Solution preparation instead FASP[2] and SR‐FASP protocolsshowed high protein identification. Both the protocols whereseen to have a bias for membrane proteins however SR‐FASPwas slightly outperforming FASP and could additionallyconvince with a better peptide coverage of the proteins.

(B) Evaluation of the cleavage performance shows an overall gooddigestion specificity with expected termini between 90‐95% onall protocols. The digestion rates were clearly superior on theFASP and SR‐FASP protocols having only 30% of under‐cleavagecompared to almost 50% of the In‐Solution and SRAD methods.

(C) Alkylation of cysteine is in all tested methods close to 100%efficiency (data not shown). A higher deamidation rate ofasparagine and glutamine might be due to the overnightdigestion at 37°C compared to a very short 15min SRA‐Digestion and an overnight RT digestion by the FASP and SR‐FASP protocols. On the other hand both FASP related methodsseem highly prone to induce oxidation of methionine whilesample handling. Overall the protocols have a similarmodification rate of about 30% except of SRAD which has only20% of modified proteins. This is supposed to be because of thevery short time of about 2h for the complete samplepreparation.

ResultsCompact and expanded cumulus cells showed very different protein expression pattern. Whilethe expanded cumulus protein expression initiation and energy metabolism as well asmembrane transportation activity was increased whereas the compact cumulus cells werecharacterized by regulatory proteins for protein expression, nucleotide metabolism andribosomal activity.

Conclusion & Outlock• The different tested sample preparation methods for low amount sample input showed 

best identification rates for both FASP methods• SR‐FASP showed slightly better performance in detection of membrane proteins• Both FASP protocols have an elevated range of oxidized methionine• SR‐FASP enabled to extract several hundreds of proteins form approx. 200 to 300 cells and 

we could discriminate between 2 biological states of the cumulus cells

• In future we are using the SR‐FASP protocol to analyze cumulus cells of different livestock to find a discriminator that allows to predict the success rate of in‐vitro fertilization

References[1] Snel et al., Nucleic Acids Res. 2000[2] Wisniewski et al, Nat Methods 2009[3] López‐Ferrer et al., J. Proteome Res, 

2005

FundingUniversity of Zurich Grant No. FK‐13‐062

Vetsuisse FacultyClinic of Reproductive Medicine

Figure 3.  (A) Overview of counted Input cells  and it’s identification results  using Scaffold (95% protein prob. 2 peptides, 95% peptide 

prob.)(B) Scaffold analysis of at least 2 fold overexpressed proteins(C) Network analysis of at least 2 fold overexpressed proteins using STRING[1]

1. Proteasome 2. Purine‐ / Pyrimidine metabolism3. Ribosome regulation

1

32

1. ATP metabolism & membr. transp. activity2. Translation initiation & Ribosomal prot.

1 2

Compact Cumulus Expanded Cumulus

A B

C

Under‐Cleavage

80%

85%

90%

95%

100%  % Non‐specific (neitherterminus expected)

% Semi‐specific (onlyone expected terminus)

% Expected termini

Over‐Cleavage

950010000105001100011500120001250013000

10001050110011501200125013001350 Protein level 1%

Global FDR

Distinct peptidelevel 1% Global FDR

Identified Proteins and Peptides

141

101

160

133

FASP

SRAD

SR‐FASP

In‐Solution

# Membrane Proteins A

B

C

0%

20%

40%

60%

80%

100%

% with substitutions

% Modified peptides

% Unmodifiedpeptides

Overall Modifications Most Frequent Modifications

0%

20%

40%

60%

80%

100%

In‐Solution SRAD FASP SR‐FASP

2

1

0

0200400600800

10001200140016001800

In‐Solution

SRAD

FASP

SR‐FASP

Counted Cells # Proteins # PeptidesCompact Cumulus 1 270 768 4485Compact Cumulus 2 270 722 4114Compact Cumulus 3 252 742 4289Expanded Cumulus 1 216 577 3291Expanded Cumulus 2 216 570 3175Expanded Cumulus 3 n/a 722 4259

Contact: claudia.fortes@fgcz.uzh.ch

• • •  www.fgcz.ch/applications/proteomics

• • •  proteomics@fgcz.ethz.ch