Efficient phosphoproteomic analysis of small amounts of biological samples (e.g. tissue biopsies) requires carefully selected enrichment and purification steps prior to the nanoflow HPLC-MS/MS analysis. Solid-phase extraction (SPE) is one of the most commonly used approaches for sample preparation. Several stationary phases are available for peptide SPE purification, however, most of the published methods are not optimized to provide good recoveries of phosphorylated peptides. Our goal was to investigate the performance of 13 self-packed and 3 commercial centrifugal SPE cartridges/spin tips, thus enhancing the efficiency of the phosphoproteomic analysis of small amounts of complex protein mixtures. Eight reversed-phase (RP), five graphite, two ion-exchange, and one hydrophilic-lipophilic balance (HLB) stationary phase were evaluated. Two RP, one graphite, and the HLB self-packed centrifugal SPE tips provided excellent results for the purification of 1 µg tissue and cell line digests. Using these methods, the sample loss was significantly reduced compared to one of the commercial SPE methods, 22-58% more unique phosphopeptides were identified, and the recovery was higher by 132-155%.

Lung cancer is the leading cause of tumor-related mortality, therefore significant effort is directed towards understanding molecular alterations occurring at the origin of the disease to improve current treatment options. The aim of our pilot-scale study was to carry out a detailed proteomic analysis of formalin-fixed paraffin-embedded tissue sections from patients with small cell or non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma). Tissue surface digestion was performed on relatively small cancerous and tumor-adjacent normal regions and differentially expressed proteins were identified using label-free quantitative mass spectrometry and subsequent statistical analysis. Principal component analysis clearly distinguished cancerous and cancer adjacent normal samples, while the four lung cancer types investigated had distinct molecular profiles and gene set enrichment analysis revealed specific dysregulated biological processes as well. Furthermore, proteins with altered expression unique to a specific lung cancer type were identified and could be the targets of future studies.

Phosphopeptide enrichment is a commonly used sample preparation step for investigating phosphorylation. TiO₂-based enrichment has been demonstrated to have excellent performance both for large amounts of complex and for small amounts of simple samples. However, it has not yet been studied for complex samples in the nanogram range. Our objective was to develop a methodology applicable for complex samples in the low nanogram range, useful for mass spectrometry analysis of tissue microarrays. The selectivity and performance of two stationary phases (TiO₂ nanoparticle-coated monolithic column and spin tip filled with TiO₂ microspheres) and several loading solvents were studied. Based on this study, we developed an effective and robust method, based on a spin tip with a non-conventional 50 mM citric acid-based loading solvent. It gave excellent results for phosphopeptide enrichment from samples containing a few nanograms of a complex protein mixture.

Genetic alterations in key oncogenes have been frequently identified in lung adenocarcinoma (LUAD), including genes encoding epidermal growth factor receptor ( EGFR ), Kirsten rat sarcoma viral oncogene homolog ( KRAS ), and anaplastic lymphoma kinase ( ALK ). In this pilot study, we aimed to characterize the differences in enriched biological pathways and phosphorylation events between LUAD tumors harboring EGFR , KRAS , or echinoderm microtubule‐associated protein‐like 4 ( EML4 )– ALK oncogenic alterations and triple wild‐type LUAD tumors (WT, without EML4–ALK , KRAS , or EGFR alterations) by mass spectrometry (MS)‐based quantitative proteomics and phosphoproteomics. We analyzed tumor regions of 82 formalin‐fixed paraffin‐embedded (FFPE) tissue sections with 6, 23, 31, and 22 samples from the EML4–ALK , EGFR , KRAS , and WT sample groups, respectively. A total of 1377 to 2189 proteins and 73 to 1781 phosphosites were quantified in these analyses. Based on the results, the samples clustered according to their genetic alteration type, and EGFR ‐mutated samples showed unique protein expression patterns. Membrane organization, vesicle organization, and vesicle‐mediated transport Gene Ontology Biological Process (GOBP) terms were significantly downregulated in EGFR ‐mutated samples compared to the other sample groups. Changes in 36 proteins and 52 phosphosites were also identified as potentially specific to a given genetic alteration. Many of these proteins have previously been linked to EGFR or KRAS mutations [e.g., cathepsin L, stimulator of interferon genes protein (STING)], whereas several phosphoproteins are associated with RNA splicing [e.g., serine/arginine‐rich splicing factor 1 (SRSF1), SRSF2, and SRSF7 proteins]. Kinase–substrate enrichment analysis indicated altered activities of 10 kinases, including mitogen‐activated protein kinases (MAPKs) and cyclin‐dependent kinases (CDKs). For example, CDK2 activity was elevated in EML4–ALK samples compared to the other sample groups. Our results could provide significant insights into further studies that could contribute to developing improved diagnostic and therapeutic strategies for LUAD.