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So, what is mass spectrometry-based proteomic profiling? How does the use of mass spectrometry distinguish this form of proteomic profiling from others? Other protein profiling methods are generally affinity based, and therefore are restricted to profiling those specific targets for which affinity ligands have been developed. Mass spectrometry-based proteomic profiling is not limited to specific targets. It can target any protein with a known sequence. And new methods enable profiling of proteins with unknown sequences. But more commonly it is run in an unbiased, untargeted manner, where the profiling is "data-dependent." This simply means that the mass spectrometer attmepts to sequence the most abundant protein first, and works its way down to lower abundance proteins as time and other instrument constraints allow.
Now, the instrument is not usually sequencing whole, intact proteins. This can be done - it is called "top-down" proteomic profiling, in contrast to the most commonly used "bottom-up," or "shotgun" proteomic profiling. But top-down methods are at a relatively early stage of development and push the limits of widely available separation, ionization and fragmentation capabilities.
Shotgun proteomic profiling measures peptides resulting from enzymatic digests of proteins in the sample. In the commonly-used data-dependent mode the instrument attempts to sequence the most abundant peptide ions available at any given point in time during the separation, and works down to lower abundance ions as time allows. In general, the more time spent on a sample, the more ions will be sequenced - up to a point. "Sequencing" constitutes fragmentation of the peptide ion in the mass spectrometer followed by computational matching of this experimentally-derived fragmentation spectrum to theoretical fragmentation spectra derived from in silico digests of known protein sequences, typically one or more proteomes. These assays yield data to include peptide sequences and peptide intensity measurments. These data can be extrapolated to infer proteins and protein intensity. A drawback of the shotgun approach is that peptides are measured, not intact proteins. So the myriad protein isoforms, also known as proteoforms, are lost if they are not identified by a peptide which is specific to that isoform.
A recent publication details the performance capabilities of the latest generation quadrupole-Orbitrap mass spectrometer from Thermo Fisher Scientific, the Q Exactive HF-X, which was recently installed in our services lab at Bioproximity. The new instrument will be the subject of an upcoming post. A highlight: the QE-HFX is capable of sequencing at a rate of up to 1200 different peptide sequences per minute and about 4000 proteins in an hour.
To wrap up: mass spectrometry-based shotgun proteomic profiling can identify and quantify peptides from any protein sample with a known genome sequence. Assays can be customized, mostly by varying assay time, to profile anywhere from a few proteins to thousands of proteins from sub-microgram quantities of protein using modern instruments. A typical workflow involves:
- protein extraction
- protein purification
- protein digestion to peptides with trypsin
- peptide purification
- UPLC-MS/MS (ultra-high performance liquid chromatography and tandem mass spectrometry)
- data processing including peptide identification, quantification and protein mapping
Each of these steps deserves a post, or two. Please subscribe to automatically receive new posts.