Proteomics Workflows
Proteomic experiments form part of an integrated set of workflows created to investigate the expression, structure and interaction of proteins in biological samples; these workflows make use of a number of different analytical procedures that can be combined in a customised fashion in order to gain maximum value from biological samples in a single experiment. Typically,
every proteomic experiment starts with a biological sample (a) that undergoes a basic extraction/purification (b) step
to produce input sample (c) for downstream analysis. The complexity of the resulting protein mixture can be reduced by an initial electrophoretic or chromatographic (d) separation of protein
components. For mass spectrometry analysis, what can follow next is the enzymatic digestion (e) of selected proteins (but also complex protein mixtures) followed by a 1D/2D (f) or higher dimension
separation of peptides via nano-LC (nano liquid chromatography). Thereafter, individual peptide fractions can be subjected to quantitative or qualitative shotgun mass spectrometry analysis to
determine the relative abundance of peptides in biological samples (g) (h), to identify individual peptides based on their m/z ratio or to generate a de-novo peptide sequence by MS/MS analysis. In
addition to the analysis of mixtures, entire tissue sections can be subjected to MALDI-TOF analysis to produce protein scans (MALDI Imaging) (I). In an alternative approach, crude protein mixtures,
fractions of protein extracts or purified single proteins can be printed on microarrays to study protein-protein, protein-molecule or protein-antibody interactions (j). Ultimately, proteins of
interest (either known or derived from mass spectrometry analysis) can be used to develop antibody affinity binding assays for biomarker validation or diagnostic purposes (k).
Glossary of Terms
1D Gel electrophoresis - The process of separating proteins based on their apparent molecular weight based on electrophoretic mobility through a gel matrix.
Chromatography - Most mass spectrometry-based experiments require proper chromatographic separation of analytes prior to carrying out an experiment. Typicall, this is achieved by using appropriate HPLC and/or nanoHPLC procedures.
Digestion - The process of enzymatic cleavage of proteins at the peptide bond to yield smaller fragments (peptides) for analysis. Usually trypsin is used for shotgun proteomics, but other enzymes such as GluC Endoproteinase, chymotrypsin and pepsin are also available for specialized analysis.
HPLC - All chromatography is based on the partitioning of analytes between two phases. Liquid chromatography, one of the most widely used separation methods, is based on the partitioning of the analytes between a mobile (liquid) phase and a stationary (solid) phase. Resolution of the analytes is achieved by exploiting a particular characteristic of the individual molecules, such as size, charge or hydrophobicity, through manipulation of mobile and stationary phase conditions. Liquid chromatography can be performed at both low pressure (LC) or at high pressure (HPLC - High Performance Liquid Chromatography).
MALDI - Matrix Assisted Laser Desorption Ionization (MALDI) is an ionization technique based in the excitation of compounds through charge transfer induced by laser energy.
Mass Spectrometry - The process of ionizing compounds followed by separation in a mass analyzer prior to detection. A variety of mass analyzers are in use and all convert the mass analysis to the mass over charge (m/z) ratio of the compound analyzed. In Proteomics, MS refers to the result of a peptide scan and MS/MS to the fragmentation of a peptide selected from the MS scan.
Middle-out proteomics - An analysis technique based on the separation of a sample first at protein level, followed by shotgun proteomics.
nanoHPLC - High pressure chromatography performed at nano-liter flow rates, typicaly 200nL/min to 300nL/min. The HPLC columns are reduced in size to 75 µm which results in extended run times of up to six hours.
PMF- Peptide mass fingerprint (PMF) analysis is based on the identification of a pure protein from a characteristic digest pattern. Protein identification relies on existing database comparisons and can be confirmed using MS/MS data. This process is not the same as (de novo) protein sequencing.
Sample enrichment- A process for selectively extracting proteins and peptides based on particular characteristics, for instance phospho-peptides.
Sample preparation - Refers to the processes used to prepare biological samples for subsequent analysis. This can include protein extraction/purification, desalting and digestion.
Shotgun proteomics - Also termed bottom-up proteomics; based on the identification of proteins from peptides in a digested complex mixture.
ToF/ToF - A Time of Flight (ToF) mass analyzer separates compounds based on their kinetic energy. The time span between entering the flight tube and detection are equated to a mass to charge ratio (m/z). A ToF/ToF analyzer contains to time of flight segments connected in series.
Top-down proteomics - Based on the analysis of proteins through peptide mass fingerprinting (PMF) and intact protein fragmentation.
