Protein Corona Gold Nanoparticles Fingerprinting Reveals a Profile of Blood Coagulation Proteins in the Serum of HER2-Overexpressing Breast Cancer Patients

Abstract

Breast cancer (BC) is a molecularly heterogeneous disease that encompasses five major molecular subtypes (luminal A (LA), luminal B HER2 negative (LB-), luminal B HER2 positive (LB+), HER2 positive (HER2+) and triple negative breast cancer (TNBC)). BC treatment mainly depends on the identification of the specific subtype. Despite the correct identification, therapies could fail in some patients. Thus, further insights into the genetic and molecular status of the different BC subtypes could be very useful to improve the response of BC patients to the range of available therapies. In this way, we used gold nanoparticles (AuNPs, 12.96 ± 0.72 nm) as a scavenging tool in combination with Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) to quantitatively analyze the serum proteome alterations in the different breast cancer intrinsic subtypes. The differentially regulated proteins specific of each subtype were further analyzed with the bioinformatic tools STRING and PANTHER to identify the major molecular function, biological processes, cellular origin, protein class and biological pathways altered due to the heterogeneity in proteome of the different BC subtypes. Importantly, a profile of blood coagulation proteins was identified in the serum of HER2-overexpressing BC patients.

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

“…The integrated peak areas were processed by MarkerView software version (AB SCIEX, USA) for a data-independent method for relative quantitative analysis [ 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. To control for possible uneven sample loss across the different samples during the sample preparation process, we performed a most likely ratio normalization [ 54 , 57 ].…”

“…In order to construct the MS/MS spectral libraries, adult and newborn (n = 6/group) were equally pooled in 3 samples. Therefore, for each developmental stage, 3 pool samples, each consisting of pooled plasma exosomes from 2 newborns or 2 adults, were run as described previously in the literature [52][53][54][55][56][57][58]. Samples were analyzed using a data-independent acquisition method, making 3 technical replicates for each sample (18 total samples: 3 replicates/sample and 3 samples/group).…”

Tear Proteomics in Keratoconus: A Quantitative SWATH-MS Analysis

“…The peptide mixtures (from sample pools) were analyzed using a data-dependent acquisition (DDA) method with micro–liquid chromatography with tandem mass spectrometry (LC-MS/MS) technology to build the MS/MS spectral libraries, as previously described. 26 – 28 Protein and peptide identification was carried out using the Protein Pilot software (version 5.0.1; Sciex; Framingham, MA, USA) with a Human Uniprot database specifying iodoacetamide as alkylation of the cysteines. The false discovery rate was adjusted to 1% for both proteins and peptides.…”

Detection of Circulating Serum Protein Biomarkers of Non-Muscle Invasive Bladder Cancer after Protein Corona-Silver Nanoparticles Analysis by SWATH-MS

“…Excess of SD and TA was removed by centrifugation at 18,840× g (×3) for 30 min. Resultant pure AgNPs were further redispersed in Milli-Q-water and the formation of the ex vivo PC was achieved following the steps shown in Figure 2 [26][27][28][29]. The characterization of the colloidal AgNPs and the formation of the PCs were confirmed by transmission electron microscopy (TEM) on a JEM 1011, JEOL instrument (Santiago de Compostela, Spain).…”

“…The combination of these nanomaterials with electrophoretic separation (SDS-PAGE) and LC-MS/MS allowed the discovery of novel serum protein biomarkers of triple-negative breast cancer (TNBC) [27]. Importantly, PC-AuNP fingerprinting revealed a profile of neutrophil-derived granule proteins in the serum of breast cancer patients [28] and, in particular, a profile of blood coagulation proteins in the serum of HER2-overexpressing breast cancer patients [29]. Molecular targets associated with thyroid tumors were also identified after the application of a similar approach for the analysis of protein extracts from thyroid tissue samples [30].…”