BottomupProteomik

Bottom-up proteomics, or Bottom-up Proteomik, is a widely used approach in proteomics in which proteins from a biological sample are enzymatically digested into smaller peptides before analysis by mass spectrometry. The resulting peptide spectra are used to identify the proteins present and to quantify their relative or absolute abundances. This approach is parallel across many samples, enabling large-scale studies of complex proteomes.

In a typical workflow, proteins are extracted, denatured, reduced and alkylated to stabilize cysteine residues, and then cleaved with a protease such as trypsin. The resulting peptides are separated by liquid chromatography and analyzed by tandem mass spectrometry (MS/MS). The acquired spectra are matched against protein sequence databases using search algorithms, yielding peptide-spectrum matches. Protein inference then aggregates peptide identifications to assign proteins, often with a false discovery rate controlled at the peptide or protein level. Quantification can be performed label-free or using isotopic labeling strategies such as SILAC, TMT, or iTRAQ.

Bottom-up proteomics offers high throughput and deep coverage for global proteome analysis and is compatible with many instrument platforms. However, it has limitations, including challenges in resolving protein isoforms and extensive proteoforms, incomplete representation of very large or hydrophobic proteins, and potential loss of information about intact protein modifications. It remains complementary to top-down proteomics, which analyzes intact proteins to preserve proteoform-level information.