Table 1.

Key proteomics techniques used for studying type 1 diabetes.

Technique	Brief description	Applications	References
Global shotgun proteomics	In global shotgun proteomics, samples are digested with proteases, most often trypsin, and the resulting peptides are analyzed by one- or two-dimensional LC-MS/MS. Peptides are identified through database search algorithms and the proteins are then inferred.	This technique is used to determine the global landscape of expressed proteins. It can be used to obtain semi-quantitative information of expressed proteins, peptides and post-translational modifications.	[15]
Isotopic labeling	Proteins and peptides can be labeled with amino acids or chemical groups containing stable, heavy isotopes. This labeling can be done in cell culture (SILAC – stable isotope labeling of amino acids in cell culture), in vivo or during sample processing.	The incorporation of heavy isotopes provides a mass shift, without altering other physical-chemical properties of peptides. Therefore, heavy labeled peptides have the same ionization efficiency and signal in the mass spectrometer, allowing samples to be multiplexed for quantitative proteomic analysis.	[16,133]
Isobaric chemical labeling	In isobaric chemical labeling, proteins or peptides are derivatized with chemical reagents (tandem mass tags – TMT, or isobaric tags for relative and absolute quantitation – iTRAQ) which incorporate a combination of heavy isotopes that provide the same intact mass. Upon tandem mass fragmentation of the labeled peptides, reporter ions of different masses are generated and facilitate peptide quantification.	Isobaric labeling is used for quantitative analysis. Commercially available kits allow to label and multiplex up to 11 samples into single analysis.	[17]
Targeted proteomics	In targeted proteomics, specific peptides are measured by selected-reaction monitoring using triple quadrupole mass spectrometers. Peptides of interest are selected in the first quadrupole, fragmented in the second and specific fragments are filtered for detection in the third quadrupole. This procedure drastically reduces the chemical background, allowing to detect trace amounts of samples. Targets are usually compared against heavy isotope-labeled peptides used as internal standards, resulting in accurate measurements of the analytes.	Precise quantification of specific proteins, peptides or post-translational modifications. This technique is especially powerful for validating targets identified by global proteomics.	[134]
Immunopeptidomics or HLA ligandome	In this approach peptides that are being presented by major histocompatibility molecules (MHC) are captured by immunoaffinity purification and analyzed by liquid chromatography-tandem mass spectrometry.	Determine the pool of antigens being presented by the organism to the immune system.	[19,20]
Immunodepletion	Highly abundant proteins from biofluids captured from samples with immunoaffinity columns. This reduces the overwhelming signals of the highly abundant proteins in the mass spectrometer, improving the detection of low abundant proteins.	Immunodepletion is a key step for deep proteomic analysis of samples, such as human blood plasma, in which the top 12 proteins represents approximately 95% of the protein mass.	[75,135]
Laser-capture microdissection	Regions of sliced tissues are precisely cut with laser.	Proteomic analysis of specific regions of tissues, such as the islets of Langerhans.	[136]
Nanoproteomics	Proteomic analysis performed in nanoscale (nanoliters of volume) to prevent sample loss.	Proteomic analysis of small samples, such as sorted cell populations or single cells.	[110,117]
Ion mobility spectrometry	Ion mobility spectrometry is a technique used to separate ionized molecules based on their mobility in an inert buffer gas under an electric field. In this technique molecules are separated by charge, size and shape.	This technique can separate isobaric molecules, allowing to characterize isomers. The separation also decreases the chemical background, enhancing the detection of analytes. Due to its separation speed, it allows samples to be analyzed in seconds, enabling analysis of thousands of samples in a single day.	[120]
Mass cytometry	In mass cytometry, cells or tissues are stained with metal-labeled antibodies, which are detected by inductively coupled plasma mass spectrometry.	Mass cytometry coupled to flow cytometry is especially powerful to determine subpopulations of cells. Imaging mass cytometry provides spatial resolution, allowing to determine the distribution of different cells in tissues.	[125,126]