**1. Introduction**

Proteomics is an important tool in the study of human biological material with the aim to extract knowledge that can improve a patients' treatment outcomes. Molecular information obtained from patient samples can be complementary to pathological observations all with the goal of faster and more accurate diagnosis, and subsequent treatment. Molecular analysis of tissue by proteomics can lead to disease classification and reveal underlying disease pathways that can further serve as a target for medical treatment.

Sample size and origin is an important aspect in sample preparation. Today, there are numerous sample preparation procedures existing which aim to improve sensitivity of detection or protein recovery from a sample. Release of proteins from native or artificial material is a crucial step in sample preparation and to improve protein recovery there are different additives such as detergents, chaotropes, buffers and salts added during the sample preparation that must be considered. Moreover, targeting special groups of proteins (e.g. membrane proteins), which are involved in key cellular functions and may be a target of pharmaceutical treatment, often represent a challenge in their isolation and analysis. Their amphipathic nature may require use of appropriate enrichment procedures all with the goal to achieve better detection.

Further, sample loss during most standard preparation procedures is inevitable, and it is even more accentuated when minute amounts of material are being processed. To minimize sample loss and thus increase sensitivity of the analysis at the MS step there have recently been several technologies developed. Specifically, improvement in technologies that allow detection of proteins down to a single cell have become available. Some of these technologies, such as nanoPOTS and micro-POTS, have already been applied to human tissues. These new possibilities to analyse small regions of tissue samples with sufficient sensitivity is opening the door to many applications such as profiling of selected regions of a tumorous zone or detection of proteins from subcellular populations. These new applications aimed at working with 1 to 100s or 1000s of cells will likely have increasing importance in clinics, but only if they can be developed into routine and robust methods.
