UBC BIOMOD 2016

MALDI-MS

September 13, 2016
Present: Ina, Tim

Task: Preliminary tests on Sigma Human Plasma APOE and synthesized DSPE-PEG-CONHNH2 using the MALDI-MS

Equipment: Applied Biosystems MALDI-TOF-MS
Note: All sample prep work was done by Nestor, from the Overall lab. APOE and DSPE-PEG-CONHNH2 were placed on ice and carried to the overall lab (LSI 4th floor)

1. Two separate samples of Sigma Human Plasma APOE were prepared:

0.75 µL of 50 µg APOE (directly from the 50 µg Sigma APOE in 50 mM ammonium bicarbonate) was combined with 0.75 µL of HCCA matrix and vortexed
- The matrix consists of 70% acetonitrile (organic solvent that will help dry the sample), 0.1% TFA (trifluoroacetic acid which will protonate sample and allow the sample to travel to the detector), and 10 mg/mL of HCCA
- Note: HCCA is standard matrix for most things, so we expected that this will give the best results
0.75 µL of 50 µg APOE was combined with 0.75 µL of 2,5-DHB—2,5-Dihydroxybenzoicacid matrix and vortexed
- The matrix consists of 70% acetonitrile (organic solvent that will help dry the sample), 0.1% TFA (trifluoroacetic acid which will protonate sample and allow the sample to travel to the detector), and 10 mg/mL of 2,5-DHB

2. Two separate samples of DSPE-PEG-CONHNH2 were prepared:

Overnight vacuum dried DSPE-PEG-CONHNH2 (~1mg) was resuspended in 1mL of dH20, and vortexed to mix.
- The sample was very stuck to the bottom of the tube, so we had to water bath sonicate the tube for around 3 mins, tap the tube several times and vortex once more
0.75 µL of the resuspended DSPE-PEG-CONHNH2 was combined with 0.75 µL of HCCA matrix (same recipe as in 1a) and vortexed
0.75 µL of the resuspended DSPE-PEG-CONHNH2 was combined with 0.75 µL of 2,5-DHB matrix (same recipe as in 1b) and vortexed

3. 1 µL of each sample was pipetted onto a MALDI plate well in the following configuration:

D6	D8	D10	D12
APOE-HCCA	APOE-DHB	DSPE-PEG-CONHNH2- HCCA	DSPE-PEG-CONHNH2- DHB

4. The MALDI plate with the samples were left to dry in the fumehood for around 2-3 mins
5. The plate was slotted into the MALDI and the following settings were observed:

D6	D8	D10	D12
APOE-HCCA	APOE-DHB	DSPE-PEG-CONHNH2- HCCA	DSPE-PEG-CONHNH2- DHB
Linear mode	Linear mode	Reflector mode	Reflector mode

Each reading took approx. 1-2 min.

6. Results were saved by Nestor and sent to BIOMOD

Notes about results:

Figure 1: APOE DHB MALDI-MS analysis showing a peak at 34.606 kDa. Sample preparation for APOE in DHB matrix yielded favourable results.

Figure 2: DSPE-PEG-CONHNH2 in HCCA matrix shows lot of variation. This is probably due to the polydispersity of the PEG2000 as each peak differs by around 40 Da. 40 Da corresponds to approximately one monomer of PEG.

Figure 3: D10_MSMS_2861.4500 - MS/MS fragmentation of the  2861.45 ion seen in Figure 2. There is a clear peak at around 607.7619 Da

October 4, 2016
Present: Ina, Lily

Task: Run the acid-cleave test on the samples. Analysis to be performed on the MALDI-MS 4700.

The samples for acid cleave were prepared in sodium acetate, which contains around 100 mM of salts.
There was difficulty in drying the samples in the hood. Nestor commented that they looked “shiny” which was not really a good sign, as they are expected to look dry and grainy after mixing the sample with the matrix and allowed to dry in the fumehood.
Unfortunately, the experiment was not finished due to a power outage in the overall lab.