Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics.

Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffin-embedded (FFPE) tissue sections that can spatially profile N-glycan compositions released from proteins in tissue-specific regions. In this study, tissue regions of interest (e.g. tumor, stroma, adipose tissue and necrotic areas) were isolated from FFPE tissue sections of advanced serous ovarian cancers (n = 3). PGC-LC-ESI-MS/MS and MALDI-MSI were used as complementary techniques to firstly generate structural information on the tissue-specific glycans in order to then obtain high resolution images of the glycan structure distribution in ovarian cancer tissue. The N-linked glycan repertoires carried by the proteins in these tissue regions were structurally characterized for the first time in FFPE ovarian cancer tissue regions, using enzymatic peptide-N-glycosidase F (PNGase F) release of N-glycans. The released glycans were analyzed by porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis. The N-glycan profiles identified by this analysis were then used to determine the location and distribution of each N-glycan on FFPE ovarian cancer sections that were treated with PNGase F using high resolution MALDI-MSI. A tissue-specific distribution of N-glycan structures identified particular regions of the ovarian cancer sections. For example, high mannose glycans were predominantly expressed in the tumor tissue region whereas complex/hybrid N-glycans were significantly abundant in the intervening stroma. Therefore, tumor and non-tumor tissue regions were clearly demarcated solely on their N-glycan structure distributions.

The importance of Mn ²⁺ for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn ²⁺ is required are yet to be fully elucidated. Here, we analyzed the effect of Mn ²⁺ limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn ²⁺ transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn ²⁺ -dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn ²⁺ -SodA and virulence-associated genes pcpA and prtA . We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC , nmlR , and czcD , in response to Mn ²⁺ stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn ²⁺ -replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn ²⁺ having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn ²⁺ transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn ²⁺ stress.

MALDI imaging mass spectrometry (MALDI-IMS) allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review.