The DAN Lab

Proteomics Protocols

Bradford Assay Protein Quantification

Notes:

  • This protein quantification method can be used for total protein content or purified proteins.
  • It is recommended to use the same buffer your proteins are stored in as your blank and dilution buffer for your sample(s) and standard curve. Alternatively, a 0.15 M NaCl solution can be used.

Materials:

Item Product Information Storage
Spectrophotometer (abs 595nm) Thermoscientific Genesys 50 (or another device capable of abs 595nm)
Bovine Serum Albumin (BSA) protein assay standards G-Biosciences, cat# 786-006 4°C
Cuvettes NA
Coomaisse Blue G-250 Thermoscientific, cat# 1856209 4°C
0.15 M NaCl solution (optional) NA

Procedure:

  1. Gently invert Coomaisse Blue dye several times to mix before use.
    1. DO NOT VORTEX
  2. Aliquot the desired amount of Coomaisse Blue in a beaker and equilibrate to RT.
    1. You will need ~7-11mL for the standard curve and 1-1.5mL per sample.
  3. Mix 20-30 µL of your desired dilution buffer with 1-1.5 mL Coomaisse Blue to create your blank.
  4. Perform a dilution scheme to create a standard curve (final volume 20-30 µL) with the following concentrations: 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/mL. Add 1-1.5 mL of Coomaisse Blue to each tube.
  5. Mix 20-30 µL of your protein sample with 1-1.5 mL Coomaisse Blue.
    1. You can also create a dilution scheme with your protein if you believe your protein concentration(s) will fall outside the linear range of your standard curve.
  6. (Optional) Incubate samples with Coomaisse Blue for 10 mins at RT.
  7. Transfer samples to cuvettes, take the OD595, prepare a standard curve plot, identify the linear range of your standard curve, and use the slope equation to quantify your proteins of interest.

Last Modified: 07/24/2021 Daven Khana

Preparation of Cell Pellet for LCMS Proteomics or Protein Extraction

Notes:

  • This protocol can be used to prepare cell pellets for LCMS proteomics or for protein extraction.
  • If using cell pellets for LCMS proteomics, this protocol should be performed as rapidly as possible to prevent changes in the proteome.

Materials:

Item Product Information Storage
Refrigerated tabletop centrifuge (capable for 10-50 mL and *1 mL) NA NA
*1X Phosphate buffered saline (PBS) See recipe for 10X Phosphate Buffer Saline RT
*1.5-2.0 mL microcentrifuge tubes Seal-Rite Microcentrifuge Tubes (USA Scientific) or comparable product NA
15-50 mL conical / falcon tubes Nunc™ Conical Sterile Polypropylene Centrifuge Tubes (ThermoFisher Scientific) or comparable product NA
10-50 mL Serological pipettes Nunc™ Serological Pipettes (ThermoFisher Scientific) or comparable product NA
*1 mL pipette NA NA
(optional) Liquid N2 or dry ice / ethanol bath NA Liquid N2 located in steel container next to cold room in Wang lab. Dry ice located in core facility.
10-50 mL bacterial culture NA NA

*Required only for LCMS proteomics

 

 

Procedure:

  1. Pre-chill all tubes and 1X PBS on ice.
    1. You’ll need 1 mL 1X PBS per cell pellet.
  2. Grow bacterial culture to appropriate OD600.
    1. For example, the typical OD600 the DAN lab uses for LCMS proteomics is ~0.45, but this is highly dependent on your experiment. For protein extractions, this OD600 can be > 1.0 (i.e., stationary phase).
  3. Using a serological pipette, collect 10-50 mL of bacterial culture in a conical tube.
    1. Ensure the culture is properly mixed before aspirating volume.
    2. It’s recommended to transfer the conical tube on ice to the centrifuge.
  4. Pellet cells:
    1. LCMS proteomics: 4000-5000 xG for 5-10 mins at 4°C.
    2. Protein extraction: 4000-5000 xG for 20-30 mins at 4°C.
  5. Aspirate or pour off supernatant.
  6. (optional) If using cell pellet for future protein extraction, flash freeze cell pellet in liquid N2 or dry ice / EtOH bath.
  7. Store cell pellet at -80°C. If using cell pellet for LCMS proteomics, proceed to step 8.
  8. Resuspend cell pellet in 1 mL cold 1X PBS. Transfer volume to a 1.5 or 2.0 mL microcentrifuge tube.
  9. Spin at max RPM for 5-10 mins at 4°C.
  10. Using a 1 mL pipette, aspirate off supernatant.
  11. (optional) Flash freeze cell pellet in liquid N2 or dry ice / EtOH bath.
  12. Store cell pellet at -80°C.

Lysis of Cell Pellet for Protein Extraction Under Denaturing Conditions

Notes

  • This protocol is adapted from Qiagen.
  • Wear proper safety gear when working with lysis buffer.

Materials:

Item Product Information Storage
Cell pellet (obtained from “Preparation of Cell Pellet for LCMS Proteomics or Protein Extraction” or similar protocol) NA NA
Lysis buffer (see below) NA 4°C
Sonicator NA 4°C room
15-50 mL conical / falcon tubes Nunc™ Conical Sterile Polypropylene Centrifuge Tubes (ThermoFisher Scientific) or comparable product NA
10-50 mL Serological pipettes Nunc™ Serological Pipettes (ThermoFisher Scientific) or comparable product NA

Lysis Buffer Composition:

Compound Amount to add (for 100 mL) Final Concentration
Urea 48.05 g 8 M
NaH2PO4 1.2 g 100 mM
NaCl 1.75 g 300 mM
Imidazole* 0.0681 g 10 mM
dH2O QS to 100 mL

*Alternatively, imidazole can be added as a concentrated solution

Lysis Buffer Preparation

  • Add ~½ the water to a beaker or graduated cylinder with a stir bar. Add the compounds and ensure they are fully dissolved. QS to 100 mL. The urea may not fully dissolve at RT, so you can heat up the solution to ~30°C to help dissolve the compound. Do NOT heat above 30°C as this may cause the urea to form cyanates, which can be detrimental for downstream protein work. Adjust to pH 8 before each use. Store in the 4°C. Currently, the longevity of lysis buffer is unknown. I have currently used it ~2 months after preparation without issue.

Procedure:

  1. Remove cell pellet (obtained from “Preparation of Cell Pellet for LCMS Proteomics or Protein Extraction” or similar protocol) from freezer storage. Thaw on ice for ~15 mins.
  2. Resuspend the cell pellet in lysis buffer.
    1. A ratio of 2 mL lysis buffer for 50 mL of pelleted culture should be sufficient, but this is dependent on cell density. For example, for coli BL21 protein expression strains grown overnight (~12 hours), this ratio is appropriate.
  3. Sonicate the cells until the solution becomes translucent, taking care to avoid foaming.
    1. Sonication Protocol: Submerge a tube rack in a bucket of ice and place your tube in the rack, ensuring the tube is secure and covered in ice. Wipe down the probe in 70% ethanol. Lower the probe into your tube until it is submerged halfway in the liquid. The probe should not be touching the sides of the tube. Repeat the following protocol until lysis is complete: 5 sec on, 5 sec off, 50% power, 2 min exposure. If foaming starts to occur, adjust the height of the probe and/or adjust the protocol.
  4. Centrifuge the lysate at 4000-5000 xG for 30 mins at RT.
  5. Collect the supernatant for protein extraction.
    1. It is recommended to save an aliquot (~20 µL) of supernatant for SDS-gel analysis to confirm that protein loss did not occur at this step.

Protein Purification of His-tagged Proteins from Cell Lysate Under Denaturing Conditions

Notes

  • This protocol is adapted from Qiagen.
  • Wear proper safety gear when working with the buffers.
  • Ensure that the centrifuge column never gets dry once the protocol begins. Liquid should always be flowing through the centrifuge column until the final elution. To ensure your washes and elutions do not mix during your collection of each flow-through, add each proximal buffer once ~95% of the preceding buffer flows through.

Materials:

Item Product Information Storage
Cell lysate (obtained from “Lysis of Cell Pellet for Protein Extraction Under Denaturing Conditions” or similar protocol) NA NA
Wash buffers, Elution buffers (see below) NA 4°C
Ni-NTA agarose slurry Qiagen 30210 or comparable product 4°C
1.5-2.0 mL microcentrifuge tubes Seal-Rite Microcentrifuge Tubes (USA Scientific) or comparable product NA
Centrifuge columns Pierce™ Centrifuge Columns, 2 mL (ThermoFisher Scientific, 89896) or comparable product NA
Ring stand and adjustable clamp NA NA
1 mL pipette NA NA
Shaker / Rocker The Belly Dancer (IBI Scientific), Fisherbrand™ Open-Air Rocking Shaker, or comparable product NA

Buffer Compositions (Wash Buffers A and B, Elution Buffers A and B)

Compound Amount to add (for 100 mL) Final Concentration
Urea 48.05 g 8 M
NaH2PO4 1.2 g 100 mM
NaCl 1.75 g 300 mM
Imidazole* 0.136a / 0.340b / 1.02c / 1.70d g 20 / 50 / 150 / 250 mM
dH2O QS to 100 mL

aWash Buffer A

bWash Buffer B

cElution Buffer A

dElution Buffer B

*The concentration of imidazole for the elution buffers can be adjusted. Alternatively, imidazole can be added as a concentrated solution

Buffer Preparation

  • If you’re preparing lysis buffer (from Lysis of Cell Pellet for Protein Extraction Under Denaturing Conditions protocol), you can just make a stock of buffer without imidazole, divide it, and add imidazole accordingly.
  • Add ~½ the water to a beaker or graduated cylinder with a stir bar. Add the compounds and ensure they are fully dissolved. QS to 100 mL. The urea may not fully dissolve at RT, so you can heat up the solution to ~30°C to help dissolve the compound. Do NOT heat above 30°C as this may cause the urea to form cyanates, which can be detrimental for downstream protein work. Adjust to pH 8 before each use. Store in the 4°C. Currently, the longevity of these buffers is unknown. I have currently used them ~2 months after preparation without issue.

Procedure:

  1. Add 500 µL Ni-NTA agarose slurry to 2 mL lysate.
    1. Thoroughly mix the Ni-NTA (without vortexing) before aliquoting.
    2. The amount of Ni-NTA agarose slurry to add can be adjusted using the above ratio based on the amount of lysate you’re working with.
  2. Lightly rock the mixture on a shaker/rocker at RT for 15-60 mins.
  3. While the lysate is incubating with the Ni-NTA, label tubes and setup protein extraction apparatus.
    1. Secure a centrifuge column to a clamp, attach the clamp to a ring stand, and adjust the height of the column so that liquid can directly flow from the outlet nozzle into a microcentrifuge tube.
    2. Remove any stored buffers from the fridge and equilibrate to RT.
  4. Following Ni-NTA/lysate incubation, lightly mix by pipetting, and add mixture to the centrifuge column, ensuring that the bottom cap is still attached. Wait ~5 mins for the layers to settle.
  5. Remove bottom cap and collect flow-through in a microcentrifuge tube.
  6. Add Wash Buffer A to the centrifuge column and collect flow-through.
    1. The amount of wash buffer to use should match the volume of lysate.
  7. Add Wash Buffer B to the centrifuge column and collect flow-through.
    1. The amount of wash buffer to use should match the volume of lysate.
  8. Elute twice with 500 µL each of Elution Buffer A and 2-4 times with 500 µL each of Elution Buffer B, ensuring to collect the flow-through for each elution.
    1. The volume of elution buffer and the number of elutions can be adjusted.
  9. Analyze initial flow-through, washes, and elutions via SDS-PAGE gel to ensure protein loss did not occur.

SDS-PAGE Gel Protocol

Notes

  • This protocol is for the Bolt Mini Gel system from Life Technologies

Materials:

Item Product Information Storage
Cell lysate (obtained from “Lysis of Cell Pellet for Protein Extraction Under Denaturing Conditions” or similar protocol) or purified protein (obtained from “Protein Purification of His-tagged Proteins from Cell Lysate Under Denaturing Conditions” or similar protocol) NA NA
Bolt Mini Gel Tank ThermoFisher Scientific A25977 Gel station
SDS-PAGE gels Bolt™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel (ThermoFisher Scientific NW04120BOX) 4°C room
SDS-PAGE running buffer 20X Bolt™ MES SDS Running Buffer (ThermoFisher Scientific B0002) 4°C room
SDS-PAGE ladder Pierce™ Unstained Protein MW Marker (ThermoFisher Scientific 26610) or comparable product 4°C
SDS-PAGE loading buffer 4X Bolt™ LDS Sample Buffer (ThermoFisher Scientific B0007) or comparable product 4°C
Gel knife ThermoFisher Scientific EI9010 or comparable product Gel station
Coomassie Blue for SDS-PAGE gels Bio-Safe™ Coomassie Stain (Bio-Rad 1610786) or comparable product 4°C
Shaker / Rocker The Belly Dancer (IBI Scientific), Fisherbrand™ Open-Air Rocking Shaker, or comparable product NA

Procedure:

  1. Prepare 450 mL of 1X running buffer per gel chamber.
  2. Place gel in the chamber and fill chamber with running buffer.
    1. Remove the white tape from the bottom of the gel.
    2. Remove plastic comb from the wells.
  3. Load ~5-8 µL of ladder.
  4. In a microcentrifuge tube or on some wax paper, mix 3 µL of loading buffer with 9 µL of protein sample and load 10 µL per well.
  5. Run the gel for ~45-60 mins / 80 V.
    1. This method can be adjusted based on the size of protein you’re looking for.
  6. Remove the gel from the chamber. Using a gel knife, carefully crack open the plastic casing.
  7. Wash the gel 3X in dH2O at RT for 5 mins each while lightly shaking / rocking.
  8. Incubate the gel with Coomassie Blue for 1 hr while lightly shaking / rocking.
  9. De-stain the gel overnight at RT in dH2

Preparation of Cell Pellet for LC-MS Based Proteomics

Notes

  • This protocol was originally provided by the Coon lab, based on their protocol “Preparation of Yeast, Bacteria, or Mammalian Cells by Lysis and Digestion for Proteomics Workflow”, version 00 prepared by Annie Jen on 20 Jan 2021.
  • Only LC-MS grade water is to be used throughout the course of the assay; otherwise, quality of the mass chromatograms and spectra may be impacted.
  • Do not mark caps with sharpie during the desalting procedure, as any errant drips may cause the sharpie material to leak into sample solution.

Objective

  • The main objective of this protocol is to provide directive on how to lyse cells and extract proteins effectively while minimizing unwanted modifications and protein degradation, for use in proteomics workflow on LC-MS

 

Materials and Equipment

 

Equivalent materials or equipment may be used unless otherwise noted.

Cell pellet (obtained from “Preparation of Cell Pellet for LCMS Proteomics or Protein Extraction” or similar protocol)
Adjustable micropipettes
Sand bath or heatblock capable of reaching 110°C
Thermometer capable of measuring up to 110°C
1.5 mL microfuge polypropylene tubes
Glass vials with cap
Microcentrifuge
Thermo Scientific™ Screw Cap Micro Tubes, 14-755-228
Thermo Scientific™ Screw Cap Microcentrifuge Tube Caps, 14-755-245
Vortexer
Centrifuge capable of vacuum drying
Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis Spectrophotometer, catalog number 840274100
pH tapes capable of reading between pH 0-3 at minimum, Millipore 1.09540.001
Conical polypropylene tubes of volumes 15 mL and 50 mL
BD PrecisionGlide™ needles, 18 G x 1-1/2 (1.2 mm x 40 mm), Sterile
Strata™-X 33 µm Polymeric Reversed Phase SPE Cartridge, 10 mg/1 mL tubes, Phenomenex, catalog number 8B-S100-AAK, do not substitute
Strata™-X 33 µm Polymeric Reversed Phase SPE Cartridge, 30 mg/3 mL tubes, Phenomenex, catalog number 8B-S100-TBJ, do not substitute
Strata™-X 33 µm Polymeric Reversed Phase SPE Cartridge, 60 mg/3 mL tubes, Phenomenex, catalog number 8B-S100-UBJ, do not substitute
Strata™-X 33 µm Polymeric Reversed Phase SPE Cartridge, 100 mg/3 mL tubes, Phenomenex, catalog number 8B-S100-EBJ, do not substitute

 

Reagents

 

Equivalent reagents may be used unless otherwise noted.

Compound CAS Number Required Grade/Purity Suggested Source/Catalog Location in DAN Lab
Water 7732-18-5 LC-MS Fisher Chemical/W6 LC-MS chemical cabinet
6 M guanidine hydrochloride solution NA NA Sigma Aldrich/SRE0066 Chemical shelf
Acetonitrile 75-05-8 LC-MS Fisher Chemical/A955 LC-MS chemical cabinet
1 M Tris buffer, pH 8.0 NA NA Thermo Fisher/15568-025 4°C
Urea 57-13-6 98% or higher Sigma Aldrich/U5378 Chemical shelf
Methanol 67-56-1 LC-MS Fisher Chemical/A456 LC-MS chemical cabinet
TFA 76-05-1 LC-MS Thermo Fisher/85183 Chemical shelf
TCEP Hydrochloride 51805-45-9 NA Sigma Aldrich/75259 4°C
2-chloroacetamide 79-07-2 NA Sigma Aldrich/22790 Chemical shelf
LysC* NA Mass Spec Grade Wako Chemicals/129-02541 -20°C
Trypsin, 0.5 µg/µL NA Sequencing Grade Promega/V5113 -80°C
Formic acid 64-18-6 LC-MS Thermo Fisher/85178 Small -20°C

*Dilute to 1 µg/µL in water and make 25 µL aliquots

Solution Preparation

 

Volumes may be scaled to suit needs of analysis.

  1. Denaturing Buffer (5.5 M Guanidine Hydrochloride in 100 mM Tris Buffer, pH ~8)
  • In a glass vial, combine 1 mL of 1 M Tris buffer, pH 8.0 with 9 mL 6 M guanidine hydrochloride
  • Mix
  1. 10X Reduction and Alkylation Buffer (100 mM TCEP, 400 mM chloroacetamide in water)
  • Weigh out 286.65 mg TCEP·HCl and 374.04 mg 2-chloroacetamide
  • Dissolve in 10 mL LC-MS grade water and mix
  • Aliquot into 5 mL microfuge tubes in 500 µL volumes and freeze.
  1. Lysis Buffer (8 M urea, 100 mM Tris, 10 mM TCEP, 40 mM chloroacetamide) – Solution must be prepared on day of use and expires day of preparation.
  • Remove a frozen 500 µL aliquot of 10X Reduction and Alkylation Buffer and allow to thaw to
  • Weigh approximately 2.40 g urea into a 5 mL conical polypropylene tube.
  • Add the thawed 500 µL 10X Reduction and Alkylation Buffer to the tube, along with 500 µL 1 M Tris buffer, pH 0.
  • QS with LC-MS grade water to 5 mL total
  • Vortex thoroughly to mix
  1. 100 mM Tris Buffer, pH ~8 – Solution must be prepared on day of use and expires day of
  • Combine 900 µL LC-MS grade water with 100 µL 1 M Tris buffer, pH 0.
  • Mix well.
  1. 10% TFA in H2O
  • While working in the hood, combine 900 µL LC-MS grade water with 100 µL
  • Mix well.
  1. 2% Formic Acid (0.2% FA)
  • Combine 10 mL LC-MS grade water with 20 µL formic acid.
  • Mix well.
  1. Elution Buffer (80% ACN, 2% TFA)
  • Combine 80 mL ACN with 20 mL LC-MS grade
  • While working in the hood, add 200 µL TFA to the solution and mix

Procedure

  1. Cell Denaturation and Lysis
  • Using a thermometer, ensure that the sand bath is at temperature 100 to 110 °C.
  • Resuspend the cell pellet in Denaturing Buffer and vortex briefly until the cell pellet is no longer
    • The resuspension volume will depend on the cell density of the cell pellet. For example, if wildtype Zymomonas mobilis cells were obtained at an OD600 of 0.45, you can start with a resuspension volume of 500µL.
  • If the concentration of the sample material is not known, perform a BCA protein assay or another protein concentration determination assay to estimate the protein concentration. Best results are achieved with protein concentration of 1-3 mg/mL; adjust sample volume if needed.
  • To a new tube, transfer enough cell material for purposes of the assay. For most label-free bottom-up proteomics experiments, 50-100 µg is enough. To fit appropriately in a 1.5 microfuge tube, it is recommended that the transferred volume not exceed 140 µL, but other tubes can be used.
    • You can dilute your samples with Denaturing Buffer so that they’re all the same concentration to make all proximal steps easier. You can store any remaining sample in the -80°C.
  • Incubate the sample in the sand bath / heat block for 5 minutes.
  • Allow sample to rest at ambient for 5
  • Incubate the sample in the sand bath / heat block for 5 minutes.
  • Add MeOH to the sample solution so that the final sample solution is 90% MeOH v/v.
  • Centrifuge samples at 9000 x g for 5 minutes. It is not recommended to centrifuge any longer, or at a faster speed, as that will complicate cell pellet resuspension.
  • With a 200 µL pipette, remove excess supernatant from the sample solution, ensuring not to disturb the cell
  • Air dry samples for 5 This can also be accomplished by placing the opened tubes upside- down on a Kimwipe.
  • Add sufficient Lysis Buffer so that the sample concentration is between 1-3 mg/mL, with a target of 5 mg/mL.
  • Vortex the sample solution for 10 minutes to resuspend the cell pellet. Sonication may be necessary if the pellet is not fully resuspended after Sample solution is now ready for digestion.

 

  1. Protein Digestion

 

NOTE: It is critical that no vortexing or violent agitation of enzymes and sample solution containing active enzymes occurs! Gently inversion or tapping is permitted, although bubble formation should be avoided.

  • Add LysC to the sample solution in the proportion of 50:1 (for example: if there is 150 µg of sample protein, add 3 µg LysC to the solution). Refer to the following equation for volume calculation:
-Volume of enzyme (µL) = Volume of the enzyme (LysC or trypsin) solution required to digest the sample solution
-Amount of sample protein (µg) = Amount of sample protein in the cell pellet, as determined by BCA if not already known
-50 = The proportion of enzyme (LysC or trypsin) to sample required to completely digest the protein without resulting in autolysis, which contaminates the sample mass spectra
-Enzyme solution concentration (µg/µL) = The concentration of the enzyme solution that will be added to the sample solution
  • Allow sample solution to incubate at ambient temperature for at least four hours. Light agitation of the sample solution can aid in the digestion process.
  • Dilute the sample solution with freshly prepared 100 mM Tris Buffer, pH ~8 so that the final concentration of urea in the sample solution is 2
  • Add trypsin to the sample solution in the proportion of 50:1. Refer to the equation in Section 2.1 for volume calculation
  • Allow sample solution to incubate at ambient temperature overnight. Light agitation of the sample solution can aid in the trypsinization process.

NOTE: If there are time constraints, there is an alternate digestion procedure that may be performed. After adding the LysC to the sample solution, allow the sample solution to incubate overnight. The following morning, add trypsin to the sample solution and allow to incubate for at least four hours prior to proceeding to Step 2.6.

  • To stop digestion, add 10% TFA solution to the sample solution until the pH is approximately 1-2. pH tape can be used to estimate the sample pH by pipetting a few µL of sample solution onto the pH tape.
  • Ensure that the centrifuge is at ambient temperature, to prevent urea from precipitating out of Centrifuge samples at 9000 x g for 5 minutes.
  1. Peptide Desalting

 

NOTE: It is recommended not to allow the cartridge solid phase to become dry while performing the desalting procedure.

  • Determine the type of desalting cartridge to be used, based on the amount of peptide in the sample, per the columns “Sample protein amount µg” and “Cartridge to be used” in Table 1.

 

Table 1. Cartridge Selection

 

Sample protein amount (µg) Cartridge to be used
≤ 500 µg 10 mg Strata X, 1 mL volume
≤ 1.5 mg 30 mg Strata X, 3 mL volume
≤ 3 mg 60 mg Strata X, 3 mL volume
≤ 5 mg 100 mg Strata X, 3 mL volume

 

  • Use a needle to pierce through the cap of a 50 mL conical tube. The conical tube will serve to hold waste Use the same needle to pierce through the cap of a screw cap micro tube with at least two holes per screw cap. The screw cap tube will be used during the final elution (Step 3.10). Do not mark the screw cap with sharpie, only mark the side of the tube. Dispose of the needle.
    • A single 50 mL conical tube can comfortably fit 3-4 needles in the lid so that you can use 1 conical tube to hold the waste for multiple samples. Waste needles can be reused for future peptide desaltings.
  • Place fresh uncapped needles through the holes that have been made in the conical and screw cap micro tubes.
  • Using the 50 mL conical tube for the initial filtering step prepared per Step 3.2, screw the cartridges onto the Loosen the cap slightly, or else flow will not occur. Label the cartridges.
  • Fill the cartridge with 100% ACN to rinse it and allow to flow to
  • Fill the cartridge with 2% formic acid and allow to flow to waste.
  • Add the digested sample material to the cartridge and allow to flow to
  • Fill the cartridge with 2% formic acid and allow to flow to waste.
  • Using the screw cap tube for the final elution prepared per Step 3.2, screw the cartridge onto the needle. Loosen the cap slightly, or else flow will not occur.
  • Fill the cartridge with the volume of Elution Buffer (µL) indicated per Table 2 and allow to flow into the screw cap micro tube.

Table 2. Elution Buffer Volume per Cartridge Type

 

Cartridge Volume of Elution Buffer (µL)
10 mg Strata X, 1 mL volume 300
30 mg Strata X, 3 mL volume 900
60 mg Strata X, 3 mL volume 1500
100 mg Strata X, 3 mL volume 2000

 

  • Remove the screw cap and dry down the desalted peptides in a vacuum Replace the screw cap with holes with a fresh screw cap. The dried desalted peptides can be frozen (-80°C) to be resuspended at a later date based on instrument availability.
    • Drying down the desalted peptides takes about < 1 hour for 300 µL.
  1. Sample Resuspension

 

  • Reconstitute sample in 0.2% formic acid so that the peptide concentration is between 1-2 µg/µL.
  • Vortex the sample solution for 10 minutes to ensure total
  • Determine sample concentration using the Thermo Scientific NanoDrop Microvolume UV-Vis Spectrophotometer or Pierce™ Quantitative Colorimetric Peptide
  • Transfer the solution into a plastic vial insert within a glass autosampler vial; avoid bubbles, especially at the bottom of the
  • Calculate the volume required to inject desired mass of peptide onto the mass spectrometer; typically 1-2 µg are used.

Last modified: 2021/12/16; DK