APAF Newsletter Vol. 2 No. 1
Welcome to APAF's Autumn 2012 newsletter.
You will be pleased to learn that APAF has now installed three TripleTOF™ 5600 mass spectrometers to provide you with unparalleled depth of peptide coverage for your proteomic analyses. For nanoLC/MS/MS applications we typically acquire 20-25 MS/MS per cycle enabling very detailed characterisation of the sample - clearly faster acquisition than competing MS instruments.
Identify novel protein-protein interactions with "Pull downs" or Co-Immunoprecipitation Mass Spectrometry
Co-Immunoprecipitation, (Co IP) is a technique used to isolate protein complexes through the use of affinity interactions. Co-IP is typically used in a serial manner to evaluate interactions one protein at a time. It requires advanced knowledge of the likely interacting partners and a useful antibody to blot for its detection. As an alternate, mass spectrometry can be used to determine the identity of co-immunoprecipitated proteins. Read more
SRM for Absolute Quantitation
Selected reaction monitoring mass spectrometry (SRM-MS) is a highly selected and sensitive method used to quantitate protein/peptide abundances in complex biological samples for targeted quantitation of proteins. SRM-MS is commonly utilised for small molecule analysis and is now developing as a mainstream technology for proteomics. Scheduled SRM-MS assays are compatible with multiplexing, enabling the quantitation for 10s-100s of analytes in a single MS run. For absolute quantitative analysis, synthetic peptides containing a stable isotopically labelled amino acid are used as standards. This enables the performance of each assay to be assessed for limits of detection/quantitation and linearity. SRM-MS assays can also be used for relative quantitation of analytes across runs.Read more
N-Terminal (Edman) sequencing
N-terminal protein sequencing continues to play a significant role in modern protein analysis and biotechnology. N-terminal sequencing is most commonly used to sequence novel proteins, confirm protein primary structure and quality (often for QC of recombinant proteins), and confirm protein N-terminus and cleavage sites. Long sequences of up to 50 amino acids are possible with this technique. APAF has worked with many groups looking at venoms and has had to develop sample preparation methods for small peptides to enable complete sequencing. Read more