INTRODUCTION Identification and characterization of low abundance proteins separated by 2D gel electrophoresis is complicated by two important factors; the use of suitable staining techniques for the visualization of the proteins on the gel and the need for sample desalting and concentration before analysis by mass spectrometry. Current silver staining protocols only allow for short development times which can lead to problems with background staining and hence problems in discerning lower abundance spots from the background. Presented in this poster is a new silver staining kit based on the method published by Sinha and co-workers (ref 1) which reduces the amount of sample handling and allows for increased development times. This reduces the amount of background staining while retaining the excellent sensitivity and mass spectrometry compatibility compared to current silver staining protocols. Once extracted, sample cleanup and preconcentration is often necessary before analysis by mass spectrometry. Techniques such as microcolumn cleanup involve intensive manual intervention and the possibility of sample loss within the column material itself. However, a new target technology recently presented (ref 2) allows for on target sample concentration and cleanup that reduces sample losses and is particularly amenable to automation. Here we show the use of this technology in the identification of low abundance silver stained gel spots not readily identifiable using standard MALDI plate technology. EXPERIMENTAL 1-D PAGE of BSA Samples BSA samples were run in multiple 26-well precast 4-2 Criterion Tris-HCl gels. Samples were prepared as serial dilutions starting with 2 ng of NEW MS COMPATIBLE SILVER STAINING/DE-STAINING AND SAMPLE CLEANUP/CONCENTRATION TECHNIQUES FOR THE IDENTIFICATION OF LOW ABUNDANT PROTEINS IN 2-D GEL SPOTS BY MALDI MASS SPECTROMETRY M.Willetts 1, M.F.Snel 1, E.Claude 1, D.C.Lee 2, D Garfin 2, J.Langridge 1, T.McKenna 1, T.Slyker 2 1 Waters Corporation, Manchester, UK. 2 Bio-Rad Laboratories Inc, CA, USA total protein load and finishing with 62.5 pg. Replicates of each sample were loaded in 3 adjacent wells in the gel. Precision Plus, unstained, protein standards were included at a 1 to 5 dilution. Following SDS-PAGE, one of the gels was stained with the Silver Stain Plus kit and the other gel was stained with the Dodeca Silver Stain kit in the Dodeca Staining system following the kit instruction protocols. 2-D PAGE of Mouse Liver Samples Using passive rehydration, 8 µg per gel of mouse liver tissue extracts were loaded into 24 cm, ph 5-8 ReadyStrip IPG strips. Following focusing in the PROTEAN IEF cell, the IPG strips were transferred to large format pre-cast 12.5 PROTEAN Plus Dodeca Cell gels. Precision Plus unstained standards were applied using precast agarose standard gel plugs. Gels stained with the Dodeca Silver Stain kit were stained using the large format Dodeca Stainer system. In addition, 2 gels were stained with two other commercially available silver stain kits according to the manufacturers instructions. Gels were imaged with a GS-8 Calibrated Densitometer. 2-D PAGE of E.coli Samples A sample of whole E.coli was separated using the same procedure as that described for the mouse liver samples above. Digestion / Cleanup The samples were processed using the Waters Micromass MassPREP Station liquid handling robot. The control software of the MassPREP station (Digest SPP 2. bis) allows destaining, reduction, alkylation, digestion, extraction and spotting of the samples onto a stainless steel target and the Waters Micromass MassPREP PROtarget Plate.
MassPrep PROtarget Plates Plate Conditioning 1 µl of acetonitrile leave for 3 seconds. 1 µl of.1 TFA (aq) leave for 2 minutes. Sample Addition Remove conditioning solution. Add 6 µl of sample in 3 acetonitrile. Allow sample to dry completely. Data Processing All data were processed and database searched using ProteinLynx Global SERVER Software 2.. During the data processing step, a near-point lockmass was applied, (Glu1)-Fibrinopeptide B. The mass spectra were smoothed, background subtracted and monoisotopic peaks were identified through the use of the MaxEntLite algorithm. Data were converted to XML format and searched against the Swiss-Prot database v4. Washing Add 5 µl of.1 TFA (aq). Wait 1 minute. Remove wash solution. Repeat 3 times. Matrix Addition Add 1 µl of matrix solution (CHCA.5 mg/ml in 9 acetonitrile /.1 TFA (aq)). Allow matrix to dry completely. Standard Target Spotting Samples were mixed 1:1 with the matrix (CHCA 2.5 mg/ml in 5 acetonitrile /.1 TFA (aq)) and 1 µl of the resulting mixture was spotted onto a standard MALDI target plate. Mass Spectrometry All data were acquired in positive ion mode on a Waters Micromass MALDI micro Mass Spectrometer. The mass spectra were collected in an automated fashion over the mass range 8-3 Da in reflectron mode. The instrument was calibrated using selected peptides from a tryptic digest of alcohol dehydrogenase. RESULTS 1-D PAGE of BSA Samples Detection sensitivity of the Dodeca Silver Stain kit was compared to the results achieved with the Bio- Rad Silver Stain Plus kit using serial dilutions of a BSA standard protein (Figure 1). Both silver stain protocols provided similar sensitivities with the detection of.25 ng of BSA protein in the gels. The Silver Stain Plus kit is a more rapid staining procedure, however the speed of the reaction prevents it from being used in the Dodeca Staining system. The Dodeca Silver Stain is completely compatible with the integrated Dodeca Staining system, which is designed to minimize handling of gels during staining, and produced gels with both excellent staining sensitivity and a reduced amount of background staining.
1 1 11 12 13 14 15 16 17 18 19 2 21 PosterREPRINT 1568.76 1881.93 188.92 1479.8 246.4 1439.81 1882.94 245.3 1569.76 1481.86 145.58 1179.6 1994.99 1415.71 247.5 1639.94 174.85 1883.95 1142.62 1249.63 248.1 1554.69 138.71 1748.76 251.6 1567.731 1 1479.793 166.14 246.15 1881.98 1163.62 1296.72 1639.926 1724.849 1 11 12 13 14 15 16 17 18 19 2 21 Figure 2. MALDI mass spectra of 4 ng BSA on gel stained with Silver Stain Plus (top) and Dodeca Silver Stain. 2-D PAGE of Mouse Liver Samples In comparing the detection results of the three silver stain kits, all showed similar detection sensitivities for the mouse liver samples as is seen in images in Figure 3. Figure 1. BSA dilution series on Dodeca Silver Stained gel (top) and Silver Stain Plus stained gel. The Dodeca Silver Stain retains the MS compatibility of the Silver Stain Plus protocol. Figure 2 shows comparison spectra from c.a. 4 ng of BSA loaded onto a 1D gel and stained with Bio-Rad Silver Stain Plus and Bio-Rad Dodeca Silver Stain. Both result in peptide coverage of c.a. 3 using peptide mass fingerprinting.
Sample Cleanup / Concentration Figure 4 shows a comparison of the MALDI spectrum obtained from spot cut from the E.coli 2D gel. No reliable hit was returned from the spectrum from the standard MALDI target plate. The spectrum from the MassPREP PROtarget shows improved signal to noise for peptides across the mass range and returned a confident hit from the database search. 1414.85 1 2212.112 1817.985 1646.97 288.355 978.548 1253.725 1941.944 2692.338 1 2212.13 288.277 1156.537 1414.816 1817.964 1646.881 194.945 1548.867 2537.163 2692.248 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 Figure 4. Comparison of mass spectra obtained from a MassPREP PROtarget (top) and standard MALDI target from a spot cut from the E.coli 2D gel CONCLUSION The Dodeca Silver Stain kit reduces background gel staining, which allows protein spots to be readily discerned while retaining the sensitivity and MS compatibility of current silver staining protocols. MassPREP PROtargets allow concentration and Figure 3. Comparison of the detection results of the Dodeca Silver Stain kit (1) and two commercially available silver stain kits (2, 3). cleanup of samples on target - enabling the identification of weaker samples not identifiable using standard MALDI target plates.
REFERENCES 1. Sinha P, Poland J, Schnolzer M and Rabilloud T, Proteomics 21, 1, 835. 2. Bouvier E, Brown J, Claude E, Gebler JL, Chen W, Gostick D, Howes K, Langridge J and Lee JP, Poster Presented at ABRF 22, Austin, Texas, USA. Sales Offices: AUSTRIA AND EXPORT (CENTRAL EUROPE, CIS, MIDDLE EAST, THE NETHERLANDS 31 76 58 72 INDIA AND INDIA SUBCONTINENT) NORWAY 47 6 384 65 43 1 877 18 7 PEOPLE S REPUBLIC OF CHINA AUSTRALIA 61 2 9933 1777 86 1 8451 8918 BELGIUM AND LUXEMBOURG POLAND 48 22 833 44 32 2 726 1 PUERTO RICO 787 747 8445 BRAZIL 55 11 5543 7788 RUSSIA/CIS 7 95 931 9193 CANADA 8 252 4752 X225 SINGAPORE 65 6278 7997 CZECH REPUBLIC 42 2 617 11384 SPAIN 34 93 6 93 DENMARK 45 46 59 88 SWEDEN 46 8 555 11 5 FINLAND 358 9 56 414 SWITZERLAND 41 62 889 23 FRANCE 33 1 348 72 TAIWAN 886 2 2543 1898 GERMANY 49 6196 46 UK 44 28 238 61 HONG KONG 852 29 64 18 HUNGARY 36 1 35 586 INDIA 91 8 837 19 IRELAND 353 1 448 15 ITALY 39 2 27 4211 JAPAN 81 3 3471 7191 KOREA 82 2 3284 13 MEXICO 52 55 5524 7636 U.S.A. AND ALL OTHER COUNTRIES: WATERS CORPORATION 34 Maple St. Milford, MA 1757 U.S.A. T: 58 478 2 F: 58 872 199 www.waters.com WATERS CORPORATION 34 Maple St. Milford, MA 1757 U.S.A. T: 58 478 2 F: 58 872 199 www.waters.com Waters, Micromass, MassPREP, PROtarget, MALDI micro, ProteinLynx and MaxEntLite are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 23 Waters Corporation Produced in the U.K. October 23 72762EN CH-PDF