pplication Note 1D PGE and Cleavable ICT Reagents Investigation of a Mammalian Cellular Model for Differential Protein Expression nalysis Using 1D PGE and Cleavable ICT Reagents Overview The use of two-dimensional gel electrophoresis combined with other analytical techniques to study differential expression has been widely employed in protein analysis. n example of which has been reported by Sullivan, et.al., using the combination of twodimensional gel electrophoresis, mass spectrometry, and Edman degradation (1) to investigate the up and down regulation of protein expression by cytokines in a human renal carcinoma cell line CHN. The invention of the isotope coded affinity tags (ICT) has offered a new approach to analyze a proteome by quantitative mass spectrometry (2). Now a combination of new cleavable ICT reagents and 1D SDS-PGE is demonstrated to be an effective alternative to previously reported approaches in the identification and differential expression of proteins from a targeted molecular weight range. The 1D PGE/cleavable ICT reagent approach for targeted protein ID and quantitation combines SDS Polycrylamide Gel Electrophoresis (PGE), isotope-coded affinity tag chemistry, and cleavable-linker technology to facilitate the identification and quantification of differentially expressed proteins. The labeled proteins, products of the labeling reactions, are separated by SDS-PGE. The targeted bands are then excised, One Dimensional SDS-PGE of CHN cell line 200kDa 116.3kDa 97.4kDa 66.3kDa 55.4kDa 36.5kDa 31kDa 21.5kDa 14.4kDa 6kDa 2.5kDa i ii iii i ii iii digested, and affinity isolated. The recovered ICT labeled peptides are used for quantification by mass spectrometry. The use of 1D PGE after ICT reagent labeling not only reduces sample complexity, but can also enrich samples of interest while removing excess reagents and placing the sample in a uniform state for subsequent proteolytic digestion. y running a Control sample, (for example, a normal cell state) and a Test sample (for example, a cytokine treated cell state), one can obtain ratios of ICT reagent labeled peptides from which protein expression levels can be determined. 200kDa 116.3kDa 97.4kDa 66.3kDa 55.4kDa 36.5kDa 31kDa 21.5kDa 14.4kDa 6kDa 2.5kDa Figure 1. Panel : Electrophoresis of the CHN cells before and after treatment with IL-4. Lane i) Mark 12 Protein Standard; Lane ii) CHN cell lysate prior to IL-4 treatment (control); Lane iii) CHN cell lysate after IL-4 treatment (IL-4 treated). Panel : Electrophoresis of the CHN cells before and after treatment with cleavable ICT reagents. Lane i) Mark 12 Protein Standard; Lane ii) Combined control and IL-4 treated samples prior to ICT labeling; Lane iii) Combined control and IL-4 treated samples after ICT labeling. The area circled was noted as an area in which differences were observed. 1 2 3 4 5 6 7 8 9 10 Key Features Designed for applications that require targeted protein identifications with quantitation for a specific molecular weight and/or known classes of proteins or complexes ccommodates complex samples that require simplification at a protein level prior to enzymatic digestion Simple convenient removal of ICT reaction contaminants and low MW by-products from the labeling reactions by SDS-PGE
Experimental Conditions In this study, CHN cells were grown with and without IL-4 treatment, harvested, washed, aliquoted (~2.5 x 10 6 cells per fraction), and suspended in 80 µl of 50mM Tris uffer, ph 8.0, containing 0.1% SDS and 0.02% Triton. The individual suspensions were lysed by placement into a sonic water bath for 30 min followed by subjection to a freeze (dry ice:acetone) and thaw cycle for three times. Cellular debris was removed by centrifugation @ 17,000 x g for 45-60 mins. Total protein concentration of each cell lysate was determined by C assay. Then, the protein concentration was adjusted to approximately 1.5 mg/ml using 50mM Tris buffer containing 0.1% SDS. pproximately 100 µg of total protein from control and IL-4 induced lysates were individually reduced with TCEP, then alkylated with cleavable ICT reagent (light) for the control and cleavable ICT reagent (heavy) for the IL-4 induced cell lysates. The control and IL-4 treated labeled samples were combined and subjected to SDS-PGE to separate proteins using a Novex 10-20% Tricine gel in a Novex XCell mini gel electrophoresis system (Invitogen life technologies). fter a brief staining (approximately 5 minutes) with Simplylue SafeStain (Invitogen life technologies) followed by destaining with water, the gel lane was segmented into 10 x 1 cm pieces as illustrated in Figure 1. The excised gel segment 8, representing the targeted MW region of ~15-30 kda, was further diced into 1 x 1 mm pieces, and then subjected to in-gel tryptic digestion overnight at 37 C. The digested peptide mixtures were extracted first by removing the supernatant and completed using 50% acetonitrile containing 0.1% TF (TF/cCN) with sonication. Expected cysteine peptides modulated in CHN cell line FPO Table 1 represents the expected cleavable ICT peptides the 8 proteins previously identified1 to be regulated by IL- 4 or IFN-. The proteins are listed as follows: 1) Glutathione S-transferase P, 2,3) Heat shock 27 kda protein (HSP 27) (pi 5.5 & 6.0), 4) Superoxide dismutase [Cu-Zn], 5) Superoxide dismutase [Mn], mitochondrial precursor, 6) Tropomyosin alpha 3 chain (Tropomyosin 3) (Tropomyosin gamma), 7) Protein Kinase C Inhibitor I. [ 8) 60S cidic Ribosomal Protein was also found in the study, but it does not have a Cys peptide to capture via this procedure.] MS/MS of isolated peptide using cleavable ICT from Superoxide Dismutase [Cu/Zn] Intensity, counts Intensity, counts 4700 MS/MS Precursor 1180.69 LCGVIEIG Mass m/2 Figure 2. Identification of Superoxide dismutase [Cu-Zn] by Panel : orthogonal QqTOF (QSTR) and Panel : LC MLDI TOF-TOF (4700 Proteomics nalyzer)
Cysteine-containing peptides were isolated by ICT Reagent Cartridge-vidin followed by TF treatment to remove the linker containing the biotin. The released peptides were analyzed for quantification and identification by LC/MS and MS/MS either using an orthogonal QqTOF (pplied iosystems QSTR System with Pro ICT Software) or LC-MLDI MS/MS (LC Packings Ulitmate LC/Proot robotic MLDI spotter and 4700 Proteomics nalyzer with GPS Explorer Software v2.0 with automated database searching (using Mascot Search Engine). For the results shown below, a molecular weight range between 15-30 kda was specifically selected as seven of the eight previously reported chemokine regulated proteins are positioned within that range. lthough samples were restricted to a defined molecular weight range, samples were not restricted by isoelectric point (pi); thus allowing removing a degree of constraint found in traditional 2D approaches. To expand protein coverage, both orthogonal QqTOF MS (QSTR System) combined with Pro ICT Software and MLDI TOF/ TOF (pplied iosystem 4700 Proteomics nalyzer) combined with GPS Explorer Software v2.0 for expression dependent analyses were used for the analyses in this application. Further integration with gene ontology information has allowed the regulated proteins to be easily classified in terms of location, biological processes, and molecular function in order to uncover some of their biological relevance in this carcinoma cell line. Results To investigate the protocol, CHN cells treated with IL-4 was used as a model system. Previous work showed that 8 proteins had various degrees of regulation in response to IL-4 or IFN-. The cysteine containing peptides (ICT Identification and quantification of TPI using results dependent analysis % Intensity % Intensity % Intensity Figure 3. Panel : MLDI MS spectrum of the spot containing triosphosphate isomerase with the 1496.8 precursor ion region expanded to visualize the ICT pair used for quantification as well as ID. Panel : MS/MS spectrum obtained from the 1496.8 precursor ion. Panel C: (see next page) The results dependent settings that were applied in obtaining the results using the 4700 Proteomics nalyzer with the inset illustrating the ICT distribution across the data set. Panel D: The results display from GPS Explorer Software v2.0 for the peptide illustrated in panel. susceptible) are listed in Table 1. Electrophoresis of the cell lines before and after treatment and cleavable ICT reagent labeling is shown in Figure 1. Mass 4700 MS/MS Precursor 1496.78 IIYGGSVTGTCK The molecular weight range between 15-30 kda was selected to pursue identification of known regulated proteins. In this example, 7 of the known proteins were known to fall in this range. Figure 2 illustrates the ID of Superoxide dismutase [Cu/Zn] by either () orthogonal QqTOF or () MLDI TOF/TOF. Glutathione S- transferase P and Superoxide dismutase [Mn], mitochondrial precursor - were also identified and quantified in this molecular weight selection (data not shown) in agreement those previously observed. In addition to the identification and relative quantification of the predicted regulated proteins by both MS platforms; it was also observed that other proteins were modulated by IL-4 treatment using an ICT reagent differential expression criteria of ± 30% from the normalized (for bias correction) H:L ratio of 1:1. In Figure 3, results
Figure 3 continued from previous page. Identification and quantification of TPI using results dependent analysis C D dependent analysis using the 4700 Proteomics nalyzer and GPS Explorer Software v2.0 found down regulation of Triosphosphate Isomerase (TPI). The advanced results dependent software allowed for expression dependent experiments using ICT reagents for targeted analysis of differentially expressed proteins (e.g. MS/MS identification of proteins that are changing upon treatment). While in Figure 4, ID and up regulation was observed for Vimentin using the QSTR System and Pro ICT Software. Combining the results from both MS platforms and their related software (orthogonal QqTOF (QSTR System and Pro ICT Software) or MLDI TOF/TOF ( 4700 Proteomics nalyzer and GPS Explorer Software v2.0)) packages, there were a total of 58 proteins identified from a total of 84 individual cysteine containing peptides. pproximately 20% were found to be classified as ribosomal proteins. While most proteins identified showed no effect by IL-4 treatment, there were 5 proteins that indicate possible cytokine regulation in this CHN cell line using an ICT reagent criteria H:L ratio of < 0.75 or > 1.25. Software integration with gene ontology information using the Celera Discovery System (CDS), a subscription-based, integrated research platform, as illustrated in Figure 5 has allowed the regulated proteins to be easily classified in terms of location, biological processes, and molecular function in order to uncover some of their biological relevance in this carcinoma cell line.
Conclusions The use of 1D SDS-PGE in combination with cleavable ICT reagents: - reduces the complexity of the sample - enriches specific areas of interest - completely removes contaminants from ICT reagent labeling - efficiently denatures proteins, i.e., better protein digestion 1D gels (as opposed to 2D PGE), combined with cleavable ICT reagents, are not restricted to pi Simplified workflow allows for parallel processing of samples prior to affinity capture Identification and quantitation of Vimentin using Pro ICT % Intensity % Intensity Figure 4. Identification and quantification of Vimentin using orthogonal QqTOF and Pro ICT software. iological information from Celera Discovery System Low abundant proteins can be identified and quantified Experiments do not require cumbersome 2D-PGE nalysis by both QSTR XL System and 4700 Proteomics nalyzer confirms and expands protein identifications and quantification from complex samples Pro ICT Software and/or GPS Explorer Software v2.0 (1st ICT reagent expression dependent software) simplifies overall data analysis Using Celera Discovery System, insight into the relationships of biological processes and molecular functions can be ascertained C References 1 Sullivan, C.M. et al., Cancer Research, 57: 1137-1145 (1997). 2 Gygi, S.P. et al., Nature iotechnology, 10:994-999 (1999). Figure 5. Current curated biological information for triosephosphate isomerase from CDS. Panel : The highlighted sections indicate the related biological processes and functions for the TPI family, SF1. Panel : The genes and transcripts plus additional evidence for the particular transcript, hct17064 (highlighted), of TPI. Panel C: The genetic information detail, including SNP s and closely related transcripts, for the Celera Transcript hct17064 (red), TPI.
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