**Abstract**

The term proteoform is used to denote all the molecular forms in which the protein product of a single gene can be found. The most frequent processes that lead to transcript modification and the biological implications of these changes observed in the final protein product will be discussed. Proteoforms arising from genetic variations, alternatively spliced RNA transcripts and post-translational modifications will be commented. This chapter will present an evolution of the techniques used to identify the proteoforms and the importance of this identification for understanding of biological processes. This chapter highlights the fundamental concepts in the field of top-down mass spectrometry (TDMS), and provides numerous examples for the use of knowledge obtained from the identification of proteoforms. The identification of mutant proteins is one of the emerging areas of proteogenomics and has the potential to recognize novel disease biomarkers and may point to useful targets for identification of therapeutic approaches.

**Keywords:** post-translational modifications, top-down, mass spectrometry, proteomic experiments, clinical application of proteoform

### **1. Introduction**

A surprise from the human genome project was the identification of 23,000 genes, far fewer than the estimated 100,000. Some events create distinct proteins that articulate various biological processes from cell signaling to genetic regulation. Thus, a single gene by allelic variations, alternative splicing and other pre-translational mechanisms, such as post-translational modifications (PTMs), conformational dynamics and functioning, may generate specific molecular forms of proteins, named "proteoforms," with different structures and different functions. Proteoforms or protein species as previously defined [1] could be identified by proteomics experiments, which include quantification of protein abundance, investigation of changes in protein expression, characterization of post-translational modifications (PTMs), identification of protein-protein interactions, a measure of isoform expression, turnover rate and subcellular localization [2]. Frequent modifications that produce proteoforms are presented in **Figure 1.**

**Figure 1.** *Types of proteoforms: RNA splicing and mutations.*
