detergentout gbs10 application note

c think proteins! think G-Biosciences! ®

Highly Efficient Detergent Removal for Improved Mass Spectrometry Analysis

Detergents are essential for protein solubility during protein extraction and sample preparation, especially when working with hydrophobic proteins. The presence of high concentrations of detergents in protein samples can impair ELISA, IEF, protease digestion of proteins and suppress peptide ionization when analyzed by mass spectrometry.

Our DetergentOUT™ GBS10 resin removes free, unbound anionic, nonionic or zwitterionic detergents (e.g. SDS, Triton® X-100 or CHAPS) from aqueous protein and peptide samples with minimal sample loss for downstream analysis by mass spectrometry (Figures 2 and 3) and other techniques. AIM

To test the DetergentOUT™ GBS10 detergent removal columns for their efficiency to remove anionic, nonionic or zwitterionic detergents (e.g. SDS, Triton® X-100 or CHAPS) from protein and tryptic peptide samples with minimal sample loss for downstream analysis by mass spectrometry and other techniques.MethodS OFFGEL Electrophoresis and SDS-PAGE

100μg samples were separated on 4-7 pH strip, 12 fractions using the 3100 OFFGEL fractionator (Agilent). After applying 20kV, fractions were collected and 1/3 was dried down and run via SDS-PAGE. The gel was stained with a fluorescent gel stain.In Solution Digestion

Samples were reduced and alkylated before digestion with Mass Spectrometry Grade Trypsin.DI-QTOF(Desorption Ionization Quadrupole Time-Of-Flight)

Samples were resuspended in 5% acetonitrile/ 0.1% trifluoroacetic acid, ziptipped using C18 resin, and infused using nanospray tips into an ABI QSTAR XL (Applied Biosystems/MDS Sciex) hybrid QTOF MS/MS mass spectrometer. TOF mass and product ion spectra were acquired using information dependent data acquisition (IDA) in Analyst QS v1.1 with the following parameters: mass ranges for TOF MS and MS/MS were m/z 300-2000 and 70-2000, respectively. Every second, a TOF MS precursor ion spectrum was accumulated, followed by three product ion spectra, each for 3 sec. LC-MS/MS

Nano-LC was performed with an nano 2D LC (Eksigent) equipped with a Dionex C18 PepMap100 column (75µm i.d.) flowing at 200nL/min. Peptides (5µl injections) were resolved on a gradient from 90.5%

solvent A (0.1% trifluoroacetic acid in MilliQ water) and 9.5% solvent B (0.1% trifluoroacetic acid in acetonitrile) to 25% solvent B in 4 minutes, then increasing to 40% solvent B over 75 minutes, and from 40-90.5% solvent B over the final 5 minutes. Mass spectrometer parameters are identical to those described above.Database Searching

The peptide tandem mass spectra were searched against NCBInr using an in-house version of MASCOT v2.2 (Matrix Science Inc). The following parameters were selected: tryptic peptides with ≤1 missed cleavage site; precursor and MS/MS fragment ion mass tolerance of 0.8 and 0.8 Da, respectively; fixed carbamidomethylation of cysteine; and variable oxidation of methionine. Data was then compiled in Scaffold (Proteome Software). Positive identification was determined based on the following criteria: ≥2 peptide sequences, minimum peptide probability of 50% and minimum protein probability of 99%.

ReSULtS ANd dISCUSSIoNDetergent Removal from Protein Samples

5µg/µl protein mixture (BSA, phosphorylase B and cytochrome C) in water was supplemented with 0.2% SDS, 0.5% CHAPS or 0.2% Triton® X-100. The samples were then treated with DetergentOUT™ GBS10, tryspin digested and analyzed by DI-QTOF (Figure 1). The results were compared to samples not treated with DetergentOUT™ GBS10. The presence of CHAPS and Triton® X-100 prevented the detection of any matching tryptic peptides and SDS inhibited the detection by ~50%. Treatment with DetergentOUT™ GBS10 successfully removed the detergent interference and the level of peptide detection was comparable to the control (Figure 1).Detergent Removal from Tryptic Peptide Samples

500µg phosphorylase B was digested with our Mass Spectrometry Grade Trypsin and the resulting tryptic peptides were treated supplemented with 0.5% CHAPS, 1% NP-40, 0.2% Triton® X-114 or 2% SDS. The samples were left untreated or passed through a DetergentOUT™ GBS10 column and then analyzed by DI-QTOF (Figure 2).

The presence of detergent in the tryptic digests prevented detection of any matching peptides, however DetergentOUT™ GBS10 treatment sufficiently removed the detergents to increase peptide recovery (Figure 2) and percentage sequence coverage (data not shown).

DetergentOUT™ GBS10 Detergent Removal Resin G-Biosciences Application Note

c think proteins! think G-Biosciences! ®

Untreated DetergentOUT™ GBS10 Treated

Control

# Peptides: 38 # Peptides: 32

0.2% SDS


0.5% CHAPS


0.2% Triton® X-100

# Peptides: 34 # Peptides: 0Figure 1: DetergentOUT™ GBS10 improves mass spectrometry spectra by removing detergents.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Conc

entr

ation

of U

nbou

nd S

DS

(mg/

ml)

Fraction #

Figure 3: DetergentOUT™ GB-S10 retains ≤6mg SDS per ml settled resin.


0

5

10

15

20

25

30

No Detergent CHAPS (0.5%) Nonidet® P-40(1%)

Triton® X-114(0.2%)

SDS (2%)

# of

Pep

tide

Spe

ctra

Det

ecte

d

DetergentOUT™ GBS10 Treated

Untreated

Figure 2: DetergentOUT™ GBS10 removes detergent and allows detection of peptide fragments by Mass spectrometry.

DetergentOUT™ GBS10 Removal of SDS2ml DetergentOUT™ GB-S10 resin was pipetted

into an appropriate column and was washed with Equilibration Buffer. To monitor SDS binding capacity, 50ml 0.1% (1mg/ml) SDS solution was continuously applied to the column. 2ml fractions were collected and assayed for the presence of SDS, using the SDS Detection & Estimation kit (Cat. No. 82021-290). Figure 3 depicts the amount of SDS detected in the flow-through, i.e. not retained by the column. The graph shows that SDS was not detected until fraction 7, so after 12mg SDS had been retained by the 2ml of DetergentOUT™ GB-S10 resin, resulting in a 6mg/ml settled resin binding capacity.

% Total Protein Recovery

Detergent % D

eter

gent

Rem

oved

BSA

Phos

phor

ylas

e B

Cyto

chro

me

C

E. c

oli L

ysat

eTriton® X-100, 2% >99 >90 >91 >92 >93Triton® X-114, 2% >96 >99 >98 >97 >91Nonidet® P-40, 1% >96 >93 >95 >91 >91Brij® 35, 1% >99 >98 >99 >97 >91SDS, 2.5% >99 >96 >97 >92 >90Sodium deoxycholate, 5% >99 >99 >99 >98 >95CHAPS, 3% >99 >92 >95 >92 >91Octyl glucoside, 5% >99 >93 >95 >96 >91Lauryl maltoside, 1% >97 >99 >99 >99 >91Tween® 20, 0.25% >98 >86 >85 >89 >85Tween® 80, 0.13% >85 >83 >81 >80 >81

table 1: A comparison of the detergent removal rates and percentage protein recovery with detergentoUt™ GBS-10. Spin columns containing 0.5ml DetergentOUT™ GBS10 resin were prepared and processed according to the protocol. 0.1ml 1mg/ml protein solutions supplemented with 1-5% detergent were processed. The DetergentOUT™ GBS10 resin effectively removed detergents with >90% protein recovery.

CoNCLUSIoNDetergentOUT™ GBS10 successful removes

anionic, non-ionic and zwitterionic detergents with minimal protein loss (Table 1). Protein samples treated with DetergentOUT™ GBS10 are suitable for mass spectrometry and other downstream applications

For polysorbate (Tween®) detergents, we recommend our DetergentOUT™ Tween® detergent removal system.

oRdeRING tABLeVWR Cat.

No. Description Sample Size (µL) Resin (µL) Size

89167-728 DetergentOUT™ GBS10-125 10-30 125 10 columns

89167-730 DetergentOUT™ GBS10-800 30-200 800 10 columns

89167-732 DetergentOUT™ GBS10-3000 200-750 3,000 10 columns

89167-734 DetergentOUT™ GBS10-5000 500-1,250 5,000 10 columns

89167-738 DetergentOUT™ GBS10 Resin - - 10 mL resin

ReFeReNCeSAlvarez, S. et al. Efficiency assay of detergent removal columns 1. on protein and peptide samples for mass spectrometric analysis. Poster presented as part of the 58th ASMS Conference on Mass Spectrometry and Allied Topics, May 23-27, 2010, Salt Lake City, Utah.Higgins, D. et al (2005) Anitmicrob. Agents Chemother. 49: 11272.


detergentout gbs10 application note

Documents