Biomarkers play an essential role in the drug discovery and development process. They provide powerful clues to genetic susceptibility, disease progression and drug response. Serum is a key source of putative protein biomarkers. One major impediment to discovering new biomarkers is the presence of salts, proteins and lipids in plasma or serum, which interferes with peptide analysis by mass spectrometry. Multiple protocols can be used to extract and enrich peptides from tissues and body fluids:
Ultrafiltration, in combination with solid phase extraction (SPE) on C18 resin is a convenient and efficient method for serum peptide purification. This approach provides more peptides for mass spectrometry analysis when compared to acetonitrile precipitation method. In addition, filter-aided sample preparation (FASP), which was originally developed using our Microcon® centrifugal filters5, has recently been optimized for use on Amicon® Ultra centrifugal filters6.
One advantage of FASP is that samples can be buffer-exchanged and concentrated through multiple spins without drying/precipitation concerns. Furthermore, FASP was adapted to 96-well plate format using our MultiScreen® Filter Plates7. Adaptation to the 96-well format permitted fast processing of biological material and opened possibilities of automating the FASP process in the future.
I. Preparation of serum peptides
II. Peptide analysis by mass spectrometry using the filtrate of Amicon® Ultra 4 mL 10kDa centrifugal ultrafilters
III. Preparation of Serum Peptides by Acetonitrile Precipitation (control samples)
Amicon® Ultra-4 30K NMWL centrifugal devices can be used for preparing serum peptides for analysis by high resolution mass spectrometry. When compared to unprocessed rat serum, data quality is improved after removing large proteins by acetonitrile precipitation (Figure 1A, B, below). The number and quality of MALDI-TOF detected peptide peaks was significantly improved using the ultrafiltered serum (Figure 1B, below).
Data quality was further improved by incorporating reverse phase chromatography in the sample preparation protocol, either alone, or in combination with acetonitrile precipitation and ultracentrifugation (Figure 2, below). Here, the ZipTip® μC18 pipette tip served as a convenient and efficient tool for micro-scale sample preparation prior to mass spectrometry. The highest signal, signal-to-noise ratio and number of detected peptides was achieved with samples prepared using all three methods (Figure 2D, below).
This approach can be used directly in combination with ZipTip® μC18 pipette tips for peptide identification by MS/MS, or as a first step prior to further surface-mediated enrichment using SELDI-TOF methods. These protocols may be applicable to other low molecular weight markers, such as drugs and metabolites.