Proteomics Training Kit

Transcription

1 CONTENTS I. INTRODUCTION II. STANDARD PREPARATION III. PROTEIN EXPRESSION: MASSPREP MIX 1 VS. MIX 2 (75 µm) IV. PROTEIN EXPRESSION: MASSPREP MIX 1 VS. MIX 2 (300 µm) V. ORDERING INFORMATION I. INTRODUCTION The Proteomics Training Standards Kit is designed to provide trainers and customers with high quality standards that can be utilized as benchmark standards for performing qualitative / quantitative proteomics experiments on their Waters UPLC inlet combined with a time-of-flight (TOF) or Q-Tof mass spectrometry detection systems. The training kit combines many of the individual standard samples that are commonly used for instrument system qualification in demonstrations and training classes, including reference compounds for calibration and lockmass. This training kit also includes stable isotope labeled (SIL) Hi3 peptide standards (PhosB and ClbB) that provide an isotopically unique exogenous standard to perform relative protein quantitation. These Hi3 SILAC peptides can be used in several different cases, including: to minimize interference with endogenous peptides, provide a second level of internal quality control (i.e., spike Hi3 SILAC standards toward the lower limit of detection), and perform relative protein quantitation for mixed proteome samples. Overall, this training kit provides the necessary foundation for LC-MS proteomic analysis and can be used to evaluate system performance and optimize methods prior to large-scale quantitative proteomics experiments. There are two versions of the Proteomics Training Standard Kits available. The Proteomics Training Standards #1 (See Table 1) contains the necessary standards for LC-MS system performance evaluation for complex proteomic digest samples. While the Proteomics Training Standards Kit #2 (See Table 2) contains standard reference peptides and proteins that are commonly used for mass spectrometry instrument specifications and/or system installation, such as [Glu1]-Fibrinopeptide B and bovine insulin. Proteomics Training Standards Kit #2 is shipped at ambient temperatures, however upon receipt of this kit, please store at -20 C to maximize long term stability.

2 Table 1. Kit Components for #1 (p/n ) Part No. Contents Component Description Amount per Vial Container vial vial x 30 ml Hi3 Peptide Standard E. coli Hi3 Peptide Standard Phos B Sodium Iodide Cesium Iodide ~ 1 nmol per peptide ~ 1 nmol per peptide 2 µg/µl 50 ng/µl vial Tryptic Digest of E. coli 0 µg/vial vial Digestion Standard Mix vial Digestion Standard Mix 2 See Table 3 in SAMPLE PREPARATION See Table 3 in SAMPLE PREPARATION vial SILAC E. coli ~ 1 nmol per peptide vial SILAC Phos B ~ 1 nmol per peptide TruView Max Recovery with non-slit 30 ml Clear Nalgene Bottle Table 2. Kit Components for #2 (p/n ) Part No. Contents Component Description Amount per Vial vial Leucine enkephalin acetate hydrate 4.0 mg (± 0.08 mg) vial [Glu1]-Fibrinopeptide B 1.0 mg (± 0.1 mg) vial Bovine Insulin 3.0 mg (± 0.15 mg) vial Horse Heart Myoglobin 3.4 mg (+1.0%, -0.0%) II. STANDARD PREPARATION Section 1. Diluent Preparation a. 97:3 water: acetonitrile + 0.1% trifluoroacetic acid (TFA) 1. Using a 0 ml graduated cylinder measure 30.0 ml of LC-MS grade acetonitrile and add to a 1 L Waters Certified Container (p/n ). 2. Add LC-MS grade or Milli-Q water to the 900 ml mark. 3. Pour contents into a 1 L graduated cylinder and add LC-MS grade or Milli-Q water to the 1.0 L mark. 5. Using caution, pour the contents of a 1.0 ml ampoule of LC-MS grade 99.5% TFA. 6. Gently swirl the solution to mix. 7. Sonicate for 5 minutes. 8. Label as 97:3 H 2 O:ACN + 0.1% TFA. 4. Pour entire contents into the 1 L Waters Certified Container and remove 00 µl of solution using a 00 µl pipette and discard the solution. 2

3 b. 75:25 water: acetonitrile + 0.1% formic acid 1. Using a 1 L graduated cylinder measure 250 ml of LC-MS grade acetonitrile. 2. Add 750 ml of LC-MS grade or Milli-Q water and fill to the 00 ml mark. 3. Transfer solution to a 1 L Waters Certified Container (p/n ). 4. Remove 00 µl of solution using a 00 µl pipette and discard the solution. 5. Add 00 µl of 99.9% LC-MS grade formic acid (FA) to the 1 L bottle. 6. Gently swirl contents or vortex to mix. 7. Sonicate for 5 minutes. d. 50:50 water: acetonitrile + 0.2% formic acid 1. Using a 0 ml graduated cylinder measure 50 ml of LC-MS grade acetonitrile. 2. Add 50 ml of LC-MS grade or Milli-Q water and fill to the 0 ml mark. 3. Transfer solution to a 250 ml bottle. 4. Remove 200 µl of solution using a 00 µl pipette and discard. 5. Add 200 µl of 99.9% LC-MS grade formic acid (FA) to the 250 ml bottle. 6. Gently swirl contents or vortex to mix. 7. Sonicate for 5 minutes. 8. Label as 50:50 H2O:ACN + 0.2% FA 8. Label as 75:25 H 2 O:ACN + 0.1% FA. c. 50:50 water: acetonitrile + 0.1% formic acid 1. Using a 0 ml graduated cylinder measure 50 ml of LC-MS grade acetonitrile. 2. Add 50 ml of LC-MS grade or Milli-Q water and fill to the 0 ml mark. 3. Transfer solution to a 250 ml bottle. 4. Remove 0 µl of solution using a 0 µl pipette and discard. 5. Add 0 µl of 99.9% LC-MS grade formic acid (FA) to the 250 ml bottle. 6. Gently swirl contents or vortex to mix. 7. Sonicate for 5 minutes. 8. Label as 50:50 H 2 O:ACN + 0.1% FA. e. 50:50 water: methanol + 1.0% acetic acid 1. Using a 0 ml graduated cylinder measure 50 ml of LC-MS grade methanol. 2. Add 50 ml of LC-MS grade or Milli-Q water and fill to the 0 ml mark. 3. Transfer solution to a 250 ml bottle. 4. Remove 00 µl of solution using a 00 µl pipette and discard. 5. Add 00 µl of LC-MS Glacial acetic acid (Fluka p/n 49199) to the 250 ml bottle. 6. Gently swirl contents or vortex to mix. 7. Sonicate for 5 minutes. 8. Label as 50:50 H2O:MeOH + 1% Acetic Acid. 3

4 Section 2. Sample Preparation a. Leucine Enkephalin (200 pg/µl) 1. Using a 5000 µl pipette, add 5.0 ml of LC-MS grade or Milli-Q water to the contents of the 4.0 mg (± 0.08 mg) bottle of leucine enkephalin. 2. Recap, shake well, and sonicate for 5 minutes. 3. Label as 800 ng/µl leucine enkephalin in water and store in the freezer (expires in 3 months). 4. Using a 0 µl pipette, transfer 50 µl of the 800 ng/µl leucine enkephalin in water to a 20-mL volumetric flask. 5. Make up to 20-mL with the 50:50 H 2 O:ACN + 0.1% FA solution and sonicate for 5 minutes. 6. Label as 2 ng/µl leucine enkephalin in 50:50 H 2 O:ACN + 0.1% FA and store in the freezer (expires in 1 month). 7. For the 200 pg/µl leucine enkephalin solution remove 2000 µl of the 2 ng/µl leucine enkephalin solution and place in a 20-mL volumetric flask. 8. Make up to 20-mL with the 50:50 H 2 O:ACN + 0.1% FA solution and sonicate for 5 minutes. 9. Label as 200 pg/µl leucine enkephalin in 50:50 H 2 O:ACN + 0.1% FA and store in the freezer (expires in 1 month).. For Fluidics systems, transfer from the 20-mL volumetric flask to a 30-mL fluidics container and label as 200 pg/µl leucine enkephalin in 50:50 H 2 O:ACN + 0.1% FA solution and store in the freezer (expires in 1 month). b. [Glu1]-Fibrinopeptide B (0 fmol/µl) 1. Using a 00 µl pipette, add 00 µl of the 75:25 H 2 O:ACN + 0.1% FA solution to the contents of the 1.0 mg (± 0.1mg) bottle of [Glu1]-Fibrinopeptide B (GFP). 2. Recap, shake well, and sonicate for 5 minutes. 3. Label as 1 mg/ml (637 pmol/µl) GFP in 75:25 H 2 O:ACN + 0.1% FA and store in the freezer (expires in 3 months). 5. Remove 78.5 µl of solution and discard. 6. Using a 0 µl pipette, transfer 78.5 µl of the 1 mg/ml (637 pmol/µl) GFP in 75:25 H 2 O:ACN + 0.1% FA solution to a 500-mL Waters Certified Container (p/n ). 7. Label as 0 fmol/µl GFP in 75:25 H 2 O:ACN + 0.1% FA and store at room temperature for use as lockmass solution. 8. Note that adding leucine enkephalin (0 fmol/µl) is recommended to perform detector voltage check and mass calibration cross validation using GFP as the TOF calibrant. c. Bovine Insulin (5 pmol/µl) 1. Using a 5000 µl pipette, add 5250 µl of LC-MS grade methanol and add 5250 µl of LC-MS grade or MilliQ water to the contents of a 3 mg (±0.15 mg) bottle of bovine insulin and sonicate for 5 minutes. 2. Label as 50 pmol/µl bovine insulin solution and store in the refridgerator (expires in 3 months). 3. Using a 00 µl pipette, transfer 2000 µl of the 50 pmol/µl bovine insulin solution to a 20-mL volumetric flask. 4. Make up to 20 ml with 50:50 water:methanol + 1% acetic acid and sonicate for 5 minutes. 5. Label as 5 pmol/µl bovine insulin soluion in 1% acetic acid and store in the refridgerator (expires in 1 month). 6. For Fluidics systems, transfer from the 20-mL volumetric flask to a 30-mL fluidics container and label as 5 pmol/µl bovine insulin solution in 1% acetic acid and store in the refridgerator (expires 1 week). 7. For LC-MS experiments, the 5 pmol/µl bovine insulin solution will need to be diluted -fold in LC-MS grade or MilliQ water to result in a 500 fmol/µl bovine insulin in a 95:5 H2O:MeOH + 0.1% acetic acid solution. 8. Inject 1 5 µl for LC-MS analysis of the intact protein, depending on the sensitivity of the instrument. 4. Using a 500-mL graduated cylinder, add 500-mL of the 75:25 H 2 O:ACN + 0.1% FA solution to a 500-mL Waters Certified Container (p/n ). 4

5 d. Horse Heart Myoglobin Solution (200 fmol/µl) 1. Using a 5000 µl pipette, add 2000 µl of 50:50 H 2 O: ACN + 0.2% FA solution to the contents of the 3.4 mg (+1.0%, -0.0%) bottle of horse heart myoglobin. 2. Recap, shake well, and sonicate for 5 minutes. 3. Label as 0 pmol/µl horse heart myoglobin solution and store in the freezer (expires 1 month). 4. Using a 5000 µl pipette, transfer 20 ml of 50:50 H 2 O: ACN + 0.2% FA solution into a 30-mL fluidics container. 5. Remove 40 µl of solution using a 0 µl pipette and discard. 6. Using a 0 µl pipette, add 40 µl of the 0 pmol/µl horse heart myoglobin solution to result in a 200 fmol/µl horse heart myoglobin solution. 7. Recap, shake well, and sonicate for 5 minutes. 8. Label as 200 fmol /µl horse heart myoglobin solution and store in the freezer (expires 1 month). 9. The 200 fmol/µl horse heart myoglobin solution will be used for direct infusion experiments using the standard ESI or nanospray ion sources.. For LC-MS experiments, the 0 pmol/µl myoglobin solution will need to be diluted -fold in LC-MS grade or MilliQ water to result in a pmol/µl horse heart myoglobin in a 95:5 H 2 O:ACN % FA solution. 11. Inject 1 µl for LC-MS analysis of the intact protein, depending on the sensitivity of the instrument. e. Hi3 Peptide Standard E. coli / Phos B (1 pmol/µl) 1. Take each vial of Hi3 E. coli ClpB / Rabbit PhosB (1 nmol/vial) and using a 00 µl pipette, add 00 µl of 97:3 H 2 O: ACN + 0.1% TFA. 2. Vortex and sonicate for 5 minutes. Stock concentration is 1 pmol/µl. 4. The 1 pmol/µl stock solutions can be diluted to the desired concentration depending on the application and column inner diameter (i.e., 75 µm vs. 300 µm chromatography). *Note that the solution is currently for 1D nanoacquity with trapping. If using a 2D RP/RP setup, change the solution to 20 mm ammonium formate ph. f. SILAC Hi3 Peptide Standard E. coli / PhosB 1. Take each vial of SILAC Hi3 E. coli ClpB / Rabbit PhosB (1 nmol/vial) and using a 00 µl pipette, add 00 µl of 97:3 H 2 O:ACN + 0.1% TFA. 2. Vortex and sonicate for 5 minutes. Stock concentration is 1 pmol/µl. 3. Label each vial as 1 pmol/µl SILAC Hi3 E. coli ClpB and 1 pmol/µl Rabbit PhosB in 97:3 H 2 O:ACN + 0.1% TFA*, respectively. 4. The 1 pmol/µl stock solutions can be diluted to the desired concentration depending on the application and column inner diameter (i.e., 75 µm vs. 300 µm chromatography). *Note that the solution is currently for 1D nanoacquity with trapping. If using a 2D RP/RP setup, change the solution to 20 mm ammonium formate ph. g. Digestion Standard Mix 1 1. Take the vial of MassPREP Mixture 1 and using a 00 µl pipette, add 00 µl of the 97:3 H2O:ACN + 0.1% TFA solution. Table 1 shows the respective pmol amounts of each protein in the vial. 2. Vortex and sonicate for 5 minutes. The stock concentrations are shown in Table Label as Mixture 1 stock solution. 4. Refer to E. coli digest Mix 1 / Mix 2 sample preparation section for dilution instructions. 3. Label each vial as 1 pmol/µl Hi3 E. coli ClpB and 1 pmol/µl Hi3 Rabbit PhosB in 97:3 H 2 O:ACN + 0.1% TFA*, respectively. 5

6 h. Digestion Standard Mix 2 1. Take the vial of MassPREP Mixture 2 and using a 00 µl pipette, add 00 µl of the 97:3 H 2 O:ACN + 0.1% TFA solution. 2. Vortex and sonicate for 5 minutes. Table 4 shows the stock concentrations of both Mix 1 and Mix Label as Mixture 2 stock solution. 4. Refer to E. coli digest Mix 1 / Mix 2 sample preparation section for dilution instructions. Table 3. Vial Contents of MDPS Mix 1 and Mix 2 protein digestion standards. Protein MDPS Mix 1 MDPS Mix 2 ADH 1.0, 50 pmol 1.0, 50 pmol GPB 1.0, 50 pmol 0.5, 25 pmol ENO 1.0, 50 pmol 2.0, 0 pmol BSA 1.0, 50 pmol 8.0, 400 pmol Table 4. Stock solution concentrations of MassPREP Mix 1 and Mix 2 protein digestion standards. Protein MDPS Mix 1 MDPS Mix 2 ADH 1.0, 50 fmol 1.0, 50 fmol III. PROTEIN EXPRESSION: MASSPREP MIX 1 VS. MIX 2 (75 µm) The following is an example of the E. coli Mix 1 vs E. coli Mix 2 protein expression experiment ran on a Waters SYNAPT G2-Si HDMS Mass Spectrometer using 75 µm scale chromatography. Section 1. Sample Preparation 1. Take the vial of lyophilized E. coli digest (0 µg/vial) and using a 0 µl pipette, add 0 µl of the 97:3 H 2 O:ACN + 0.1% TFA solution. 2. Label as 1 µg/µl E. coli stock solution. 3. In a TruView LC-MS Certified Total Recovery vial with a pre-slit PTFE/silicone septa (p/n CV) add the following solutions and volumes defined in Table Table 6 shows the resulting concentrations of the MassPREP Mix 1 / Mix 2 proteins and their expected molar ratios. Table 5. Sample preparation details for E. coli Mix 1 / E. coli Mix 2 for 75 µm scale chromatography. *Note that the SILAC Hi3 standards are added for internal quality control (QC) standard. Adding these SILAC standards are optional. E. coli Mix 1 Solution E. coli stock digest (1 µg/µl) Volume (µl) E. coli Mix 2 Solution E. coli stock digest (1 µg/µl) Volume (µl) Mix 1 stock 20 Mix 2 stock 20 GPB 1.0, 50 fmol 0.5, 25 fmol ENO 1.0, 50 fmol 2.0, 0 fmol *SILAC Hi3 E. coli ClpB fmol/µl *SILAC Hi3 E. coli ClpB fmol/µl BSA 1.0, 50 fmol 8.0, 400 fmol *SILAC Hi3 PhosB 50 fmol/µl *SILAC Hi3 PhosB 50 fmol/µl 97:3 H 2 O:ACN + 0.1% TFA 50 97:3 H 2 O:ACN + 0.1% TFA 50 Table 6. Resulting concentrations of the MassPREP Mix 1 and Mix 2 proteins after dilution into the E. coli lysate background. Protein MDPS Mix 1 MDPS Mix 2 ADH 1.0, fmol 1.0, fmol GPB 1.0, fmol 0.5, 5 fmol ENO 1.0, fmol 2.0, 20 fmol BSA 1.0, fmol 8.0, 80 fmol 6

7 Section 2. LC-MS/MS (75 µm) Experimental Conditions Conditions: Inlet: Solvent A: Solvent B: Weak wash: Trapping: nanoacquity System ran in a single pump trapping mode 0.1% formic acid in water 0.1% formic acid in acetonitrile 97:3 H 2 O:ACN + 0.1% TFA µl/min for 3 min, µl loop Column: ACQUITY ULC M-Class HSS T3 Column, 1.8 µm, 75 µm x 150 mm, 1/pkg (p/n ) Trap: ACQUITY UPLC M-Class Symmetry C 18 Trap Column, 0A, 5 um, 180 um x 20 mm, 2G, V/M 1/pkg (p/n ) Figure 2. Progenesis QI for Proteomics: Data was processed for the quintuplicate injections of E. coli Mix 1 and E. coli Mix 2. The image illustrates the Protein Statistics data review with a Principal Component Analysis showing each injection for the relative expression levels of ALBU_BOVIN (expected expression level increase 8-fold). Gradient profile: Time Flow Rate (min) (µl/min) %A %B Curve Figure 3. Protein expression comparison of the relative fmol amounts of each MassPREP Mix 1 and Mix 2 standard protein (75 µm chromatography). Note that the average fmol amounts are shown with the respective standard deviation error bars. Figure 1. Base Peak Intensity (BPI) chromatograms: (A) E. coli Mix 1 and (B) E. coli Mix 2 ran using a 75 µm x 150 mm column. MS conditions: MS E using alternate scanning low (4 V) and elevated energy (15 45 V) and a scan time of 0.5 seconds. Injection volume: 1.0 µl of the sample for each condition. IV. PROTEIN EXPRESSION: MASSPREP MIX 1 VS. MIX 2 (300 µm) The following is an example of the E. coli Mix 1 vs E. coli Mix 2 protein expression experiment ran on a Waters SYNAPT G2-Si HDMS Mass Spectrometer using 300 µm scale chromatography. The ACQUITY UPLC 2D M-Class HCP System was used for these experiments. 7

8 Section 1. Sample Preparation 1. Take the MassPREP Mixture 1 / Mixture 2 stock solutions prepared earlier and dilute both Mix 1 / Mix 2 according to the following procedure. 2. Using a 0 µl pipette, add 200 µl of Mix 1 and Mix 2 and place the volume in two individual TruView Max Recovery vials and label as Mix 1 and Mix 2, respectively. Section 2. LC-MS/MS (300 µm) Experimental Conditions Conditions: Inlet: 2D ACQUITY UPLC M-Class HCP System ran in simulated 1D mode Solvent A: 0.1% formic acid in water Solvent B: 0.1% formic acid in acetonitrile 3. Using a µl pipette, add 1 µl of the 1 pmol/µl SILAC E. coli ClpB to each Mix 1 and Mix 2 sample vials. 4. Using a µl pipette, add 5 µl of the 1 pmol/µl SILAC Rabbit PhosB to each Mix 1 and Mix 2 sample vials. 5. Using a 00 µl pipette, add 794 µl of 20 mm ammonium formate ph to each Mix 1 and Mix 2 sample vials. 6. Cap each vial and vortex. 7. Label these solutions as Mix 1 Diluted Solution and Mix 2 Diluted Solution. These solutions will be used to resuspend lyophilized aliquots of E. coli digest. Table 7 contains the diluted concentrations of Mix 1 and Mix Using a 0 µl pipette, add 0 µl of Mix 1 Diluted Solution to a vial of E. coli digest (0 µg). Cap the vial and Vortex. 9. Using an additional vial of E. coli digest (0 µg), add 0 µl of Mix 2 Diluted Solution. Cap the vial and Vortex. Note that for the 300 µm or 1 mm scale chromatography it is recommended that a least two vials of E. coli digest be used for the E. coli Mix 1 vs. E. coli Mix 2 experiment. Trapping: Column: 2nd dimension: Trap: Gradient profile: Simulated 1D (elute from 1st dimension with 50% B) µl/min for 26 min, 0 µl loop 1st dimension: XBridge C 18, 2.5 µm, 1.0 x 50 mm (p/n ) ACQUITY UPLC M-Class HSS T3 Column, 1.8 μm, 300 μm x 150 mm (p/n ) ACQUITY UPLC M-Class Symmetry C18 Trap Column, 0A, 5 μm, 300 μm x 25 mm, HCP (2D) (p/n ) Time Flow Rate (min) (µl/min) %A %B Curve Table 7. Diluted concentrations of MDPS Mix 1 and Mix 2 protein digestion standards for 300 µm chromatography. Protein MDPS Mix 1 MDPS Mix 2 ADH 1.0, fmol 1.0, fmol GPB 1.0, fmol 0.5, 5 fmol ENO 1.0, fmol 2.0, 20 fmol BSA 1.0, fmol 8.0, 80 fmol Figure 4. Base Peak Intensity (BPI) chromatograms: (A) E. coli Mix 1 and (B) E. coli Mix 2 ran using a 300 µm x 150 mm column. MS conditions: MS E using alternate scanning low (4 V) and elevated energy (15 45 V) and a scan time of 0.5 seconds. Injection volume: 2.0 µl of the sample for each condition. 8

9 Figure 5. Progenesis QI for Proteomics: Data was processed for the quintuplicate injections of E. coli Mix 1 and E. coli Mix 2. The image illustrates the Review Proteins data review section with a list of the protein identifications and the relative fold change amounts and Hi3 relative quantitative fmol amounts for each protein identification. The highlighted protein, ENO1_YEAST, shows an increase in the expression level for E. coli Mix 2 sample (expected expression level increase 2-fold). Figure 6. Protein expression comparison of the relative fmol amounts of each MDPS Mix 1 and Mix 2 standard protein (300 µm chromatography). Note that the average fmol amounts are shown with the respective standard deviation error bars. 9

10 V. ORDERING INFORMATION Proteomics Training Standards #1 (p/n ) Includes the following p/n s to be stored at room temperature: Part No. Contents Component Description Amount per Vial vial Hi3 Peptide Standard E. coli ~ 1 nmol per peptide vial Hi3 Peptide Standard Phos B ~ 1 nmol per peptide * 1 x 30 ml Sodium Iodide Cesium Iodide 2 µg/µl 50 ng/µl vial Tryptic Digest of E. coli 0 µg/vial vial Digestion Standard Mix 1 ~ 1 nmol vial Digestion Standard Mix 2 ~ 1 nmol vial SILAC E. coli ~ 1 nmol per peptide vial SILAC Phos B ~ 1 nmol per peptide Proteomics Training Standards #2 (p/n ) Includes the following p/n s to be stored at -20 C ± C (frozen) immediately upon arrival: Part No. Contents Component Description Amount per Vial * 1 vial Leaucine Enkephalin 4.0 mg * 1 vial [Glu1]-Fibrinopeptide B 1.0 mg * 1 vial Bovine Insulin 3.0 mg * 1 vial Horse Heart Myoglobin 3.4 mg *Not sold separately outside of the kit. Waters, The Science of What s Possible, ACQUITY UPLC, Xevo, SYNAPT, nanoacquity, and Progenesis are registered trademarks of Waters Corporation. MassPREP, and TruView are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed/Produced in the U.S.A. September EN IH-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: