HeLa quality control sample preparation for MS-based proteomics

doi:10.21203/rs.3.pex-2155/v1

Method Article

HeLa quality control sample preparation for MS-based proteomics

https://doi.org/10.21203/rs.3.pex-2155/v1

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Cell lysis and digestion protocol of HeLa cell line for quality control measurments of mass spectrometry-based proteomics measurments.

In solution digest of human proteome sample for HeLa quality control runs of mass-spectrometers used at Novo Nordisk Foundation Center for Protein Research. The protocol has no specific author and is adapted by individual researchsers at the institute.

6M Guanidine Hydrochloride (MW: 95.53g/mol) – 5.73g

5mM TCEP (stock: 0.5M) – 100x – 100ul

10mM CAA (stock: 550mM) – 55x – 182ul

100mM Tris pH 8.0 (stock 1M) – 10x – 1ml

2ml of Bradford

Cell lysis

Prepare 10ml Lysis buffer fresh

6M Guanidine Hydrochloride (MW: 95.53g/mol) – 5.73g

5mM TCEP (stock: 0.5M) – 100x – 100ul

10mM CAA (stock: 550mM) – 55x – 182ul

100mM Tris pH 8.0 (stock 1M) – 10x – 1ml

Water ~ 5ml

Heat lysis buffer to 99°C
IF STARTING FROM PELLET – SKIP STEP 4 and 5.
Aspirate media completely from the plate
Wash with 10 ml of PBS twice
Add 1000ul lysis buffer (and scrape cells off the plate) and collect in a 15 ml falcon tube
Heat lysate at 99°C for 10min in the heating block while shaking
Sonicate with microtip probe for 2min with pulses of 1sec ON, 1sec OFF at an amplitude of 50%.

Measure protein concentration using Bradford Dye Reagent (in common fridge)

Prepare protein standards in the range 0, 1, 2, 4 and 8ug/ml using BSA
Mix 1ml of Bradford and the appropriate volume of BSA (stock: 1mg/ml)
Mix 1ml of Bradford and 1ul of your protein sample
Prepare a blank sample with 1ul lysis buffer
Vortex the samples gently and incubate for 5min
Measure the absorbance at 595nm for the standards and the sample
Calculate the protein concentration by plotting a standard curve of the absorbance vs. protein sample concentration

Digestion

Digest lysate with LysC: protein ratio 1:100 (w/w) (stock: 0.5ug/ul) and incubate shaking at 25°C for 60min (O/N) (5000ug = 100ul)
Dilute to minimum 2M Guanidine hydrochloride with 25mM Tris pH 8.5 (40x from stock)
Digest with trypsin: protein ratio 1:100 (w/w) (stock: 0.5ug/ul) and incubate at 37°C shaking O/N at 300rpm (5000ug = 100ul)
Stop the digest by quenching with TFA (pH should be reduced to ~2 by adding 1/10 volume of 10% TFA) Check with pH strip (NB: chemical hood!!)
Upconcentrate on Sep-Pak

Some former researchers to establish the protocol over a longer period of time.

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HeLa quality control sample preparation for MS-based proteomics

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