A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.
A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns.
AIMMS Division of BioMolecular Analysis, VU University, Amsterdam, The Netherlands.
Linda
L
Switzar
Jordy
J
van Angeren
Martijn
M
Pinkse
Jeroen
J
Kool
Wilfried M A
WM
Niessen
D053000:Analytic Sample Preparation Methods / Q000295:instrumentation*; D002457:Cell Extracts; D002477:Cells / Q000378:metabolism*; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D036103:Nanotechnology; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*
D002457:Cell Extracts; D010455:Peptides; D011506:Proteins
Bottom-up proteomics; Cellular proteomics; Filter plate; High-throughput sample preparation; LC-MS; Technology