The first stage of a bottom-up proteomic workflow is to obtain a protein mixture from biological samples. This includes sample pretreatment, enzyme inhibition, homogenization, protein extraction/precipitation, and protein fractionation. Sample preparation is crucial in proteomics analysis and the procedure vary depending on the sample type.

Cell samples

It is necessary to break down the cell membranes and homogenize the samples with lysis buffer and sonication. Enzymes that may affect the protein structure, such as proteases and phosphatases, should be inhibited by maintaining the samples at a low temperature and adding an enzyme inhibitor cocktail. Typical protease inhibitors include pepstatin A, leupeptin, aprotinin, and chymostatin. Phosphatase inhibitors, including sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and beta-glycerophosphate, are used to protect phosphorylated proteins. These enzyme inhibitors are usually added to the lysis buffers prior to sonication.

To the homogenized samples, organic solvents (such as acetone, methanol, or ethanol) and their mixtures with trichloroacetic acid or sodium deoxycholate are added to precipitate the proteins. The protein pellets are then collected and washed with pre-chilled solvent to remove contaminants.

Biological fluids (blood, urine, saliva, nasal fluids, tears, and aqueous humor)

Generally, biological fluids are first centrifuged to remove debris and then diluted with an appropriate amount of buffer to facilitate the subsequent enzymatic digestion process.

In the case of urine samples, proteins are extracted using solvent precipitation, ultrafiltration, centrifugal filtration, dialysis, and lyophilization.

Blood samples are usually centrifuged to collect plasma or serum. Plasma and serum are then subjected to immunodepletion to remove high-abundance proteins to allow the identification of low-abundance proteins.


Tissue samples are first rinsed with ice-cold saline to remove blood, serum, and fat and homogenized subsequently using different apparatuses, such as a pestle and mortar, mechanical rotor–stator grinders, bead-beating homogenizer, and sonication.

Liquid nitrogen pulverization involves the use of liquid nitrogen to freeze tissues for a short time. It can preserve protein integrity without generating heat. The frozen tissues are then disrupted using a pestle and mortar, a pulverizer or a pressure cycling homogenizer (PCT)for tissue disruption.  The homogenized tissues can then be subjected to a protein extraction step to obtain samples with high protein concentrations.