Sample preparation

How to prepare your samples for MassSpecPreppy.

Introduction

Sample preparation is the key to a successful experiment
  1. If you have a insufficient sample disruption the ID number might be hampered
  2. If DNA is still present in your sample the efficient binding of proteins to the SP3 beads is jeopardized because DNA can bind to the beads as well! In addition, LC-MS columns might be damaged because of the DNA contamination.
  3. Insufficient inactivation of proteases in your sample will lead to uncontrolled protein cleavage. This must be minimized.

To tackle all these issues please follow carefully the protocol below

Cell disruption and protein lysate generation

buffers
buffer composition
sample buffer 20mM HEPES, 1% (w/v) SDS, pH8
wash buffer 20mM HEPES, pH8
Benzonase cofactor solution 1M MgCl2
Benzonase dilution buffer 20 mM HEPES pH8, 20 mM NaCl, and 2 mM MgCl2
tips for different samples
  1. Bacterial sample: For a bacterial cell pellet use 16 OD-units (e.g. 16 ml of a culture at OD = 1 should be harvested or 32 ml of a culture at OD = 0.5). This is important to keep the samples in the range of the capacity of the used buffers and to equalize your samples.
  2. Human plasma: Dilute human plasma 1:50 in SDS containing buffer (e.g. 20mM HEPES, 1% (w/v) SDS, pH8) and inactivate it at 95°C for 5 minutes

bacterial or solid samples

  • harvest the needed amount of material
  • wash the pellet 2 time with 1 ml wash buffer
  • discard the supernatant and resuspend the pellet in 100µl sample buffer
  • immediately disrupt the cells in the dismembrator (ball mill) for 3 min at 2600 rpm
Tip

in a Teflon vessel on liquid nitrogen with an 8 mm diameter steel ball

  • resuspend the cell powder in 400 µl of pre-heated (95°C) sample buffer and transfer the viscous lysate into a fresh 1.5 mL low bind pre-lubricated reaction tube and shake for 1 min at 95°C at 1400 rpm
  • then put the lysate on ice and add 2 µL of Benzonase cofactor solution (1 M MgCl2) (final 4 mM MgCl2)
  • now add 1 µL of a 1:100 diluted Benzonase (Pierce Universal Nuclease No#88702; 250 U/µl) stock solution (2.5 U/µL) and mix by short vortexing
  • incubate at 37°C for 15 minutes
  • now put the samples in the ultrasonic bath for 5 minutes the viscous lysate should liquefy by complete degradation of DNA and RNA
Tip
  • Benzonase protein should then always be included in the contaminant list of the organism specific library fasta-file
  • Benzonase should be diluted for easy handling of small quantities with 5 mM Tris-HCl pH 7.4, 20 mM NaCl, and 2 mM MgCl2
  • Diluted samples can be stored at 4°C for several days without loss of activity
CAUTION

PBS buffer (phosphate buffered saline) inhibits the Benzonase activity

  • now put the samples in the ultrasonic bath for 5 min
Note

The viscous lysate should liquefy by complete degradation of DNA and RNA. This step ensures solubilization of all DNA and RNA binding proteins and avoids the binding of long DNA or RNA stretches to the SP3 beads.

  • centrifuge the samples for 30 min at 17000xg at room temperature
  • transfer the protein lysate into a fresh 1.5 mL low bind pre-lubricated reaction tube, store or dispose the residual cell debris
      1. a bacterial lysate should now have a concentration between 1.5 and 3 µg/µL (important for microBCA protein concentration measurement)
Note
  • store the raw lysates at -80°C
  • for thawing place samples to 65°C

blood plasma samples

buffers
buffer composition
sample buffer 20mM HEPES, 1%SDS, pH8
  • dilute sample blood plasma with 1x sample buffer (human plasma 1:50)
  • incubate 5 minutes at 95°C to inactivate the plasma
  • store the samples at -80°C
  • for thawing place samples to 65°C

protein concentration determination using microBCA

buffers
buffer composition
1x sample buffer 20mM HEPES, 1%SDS, pH8
2x sample buffer 40mM HEPES, 2%SDS, pH8
microBCA working reagent 25:24:1 (MA:MB:MC) mixture
  • follow the guidelines of MassSpecPreppy