4
Protein ID Solutions Sample Prep Guide
1
Overview of Sample Preparation Steps with the Protein ID Solutions
Digestion
The next part of the workflow centers around protein digestion. While it is
possible to analyze intact proteins by mass spectrometry, this type of analysis
usually provides only a molecular weight. When the proteins are digested with
site-specific proteases, and the peptides are analyzed by LC/MS, the analysis
produces a much more informative peptide map. The peptide map gives the
molecular weights of all the peptides. The peptides themselves can be
fragmented in MS/MS experiments, where they cleave between amino acids.
The MS/MS spectra yield sequence information that can be searched against
DNA and protein databases.
This part of the workflow also includes reduction and alkylation. It is common
practice to reduce and alkylate cysteine residues prior to digestion. Reduction
breaks existing disulfide bonds and alkylation prevents the cysteines from
reacting to form new disulfide bonds. Reduction and alkylation have three
major benefits:
· They increase the efficiency of digestion and reduce the number of missed
cleavages.
· They simplify peptide maps by removing disulfide linkages.
· They prevent formation of new disulfide linkages at the high pH typically
used for digestion.
The primary disadvantages are potentially reduced recoveries and the
possible introduction of contamination.
Cleanup
The next part of the workflow is sample cleanup. This step removes salts and
other contaminants that interfere with analysis by mass spectrometry. For the
Nanospray and HPLC-Chip/MS Protein Identification Solutions, Cleanup C18
Spin Tubes or Cleanup C18 Pipette Tips are used as a convenient means to
remove contaminants that could suppress ionization or contribute noise to MS
analyses. The pipette tips are used for smaller amounts of protein, such as
from 2D gel spots, while the spin tubes are used for greater sample loads. The
steps after sample cleanup are covered in the LC/MS Application Guide.
Summary :
4 Protein ID Solutions Sample Prep Guide 1 Overview of Sample Preparation Steps with the Protein ID Solutions Digestion The next part of the workflow centers around protein digestion. For the Nanospray and HPLC-Chip/MS Protein Identification Solutions, Cleanup C18 Spin Tubes or Cleanup C18 Pipette Tips are used as a convenient means to remove contaminants that could suppress ionization or contribute noise to MS analyses.
Tags :
protein,digestion,sample,cleanup,used,disulfide,workflow,part,analysis,alkylation,reduction,peptide,peptides