MALDI-MS Protein Profiling of Chemoresistance in Extracellular Vesicles of Cancer Cells.

Cancer cells communicate with the whole organism via extracellular vesicles (EVs), which propagate molecular information in support of the malignant phenotype. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was employed for protein profiling of EVs derived from CCL-228 as the primary colon tumor, the lymph node metastasis CCL-227, and subclones resistant to 5, 25, and 125 µM 5-fluorouracil (FU). EVs were harvested from cell culture supernatant by ultracentrifugation to serve as a model for circulating cancer cell-derived biomarker carriers from body fluids (i.e., liquid biopsy). Protein mass spectra were recorded using standard MALDI matrixes (e.g., CHCA, sinapinic acid) in the range m/ z 2000-20000 on different MALDI-TOF-MS systems and subjected to multivariate data analysis . By using hierarchical clustering, PCA and PLS-DA, discriminatory protein patterns of the EVs from the different cell populations were obtained. Peaks in the range  m/ z 2000-6500 and m/ z 5500-15500 were found to be unique to EVs and the cells, respectively. This clearly demonstrates the differential expression of proteins in EVs as the result of an increasing chemoresistance of their parent cells. The sensitivity of the MALDI-MS based assay was in the low µg/mL (≈1.2-5 × 1010 particles/mL) range. Consequently, our MALDI-MS protein profiling approach shows the potential to serve as novel tool for minimally invasive cancer diagnostics and chemotherapy monitoring in the future, e.g., for early detection of therapy resistance without biopsy.

In-source decay and pseudo tandem mass spectrometry fragmentation processes of entire high mass proteins on a hybrid vacuum matrix-assisted laser desorption ionization-quadrupole ion-trap time-of-flight mass spectrometer.

In-source decay (ISD), although a process known for decades in mass spectrometry, has a renewed interest due to increased theoretical knowledge in fragmentation processes of large biomolecules coupled with technological improvements. We report here an original method consisting of isolating matrix-assisted laser desorption ionization (MALDI)-generated in-source fragments of large proteins and subsequently performing selective fragmentation experiments (up to four cycles) using a hybrid MALDI quadrupole ion-trap time-of-flight mass spectrometer (MALDI-QIT-TOF). This technology takes advantage of keeping high resolution on the selection of precursors and detection of fragments. It allows exhaustive N- and C-terminal sequencing of proteins. In this work, human serum albumin (HSA), ß-casein, and recombinant Tau proteins were submitted to in source decay in the MALDI source. The fragments were stored in the ion-trap and submitted to sequential collision-induced dissociation (CID). Finally, ISD and pseudo MS(n) were performed on oxidized Tau protein and acetylated bovine serum albumin to identify amino acid modifications. This work highlights the potential of the MALDI-QIT-TOF instrument for pseudo MS(n) strategies and top down proteomics.

Sequencing of T-superfamily conotoxins from Conus virgo: pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry.

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

Investigation of the electrospray response of lysine-, arginine-, and homoarginine-terminal peptide mixtures by liquid chromatography/mass spectrometry.

Guanidination has been used to investigate how modification of the lysine eta-amino group into the corresponding guanidino group affects response in electrospray (ES) mass spectrometry (MS). Selected ion monitoring (SIM) analysis of equimolar mixtures containing arginine-, lysine- and the corresponding homoarginine-terminal peptides following liquid chromatography (LC) showed differences in ES response. The ionisation behaviour of the standard peptides is in accordance with the postulated higher stability of the guanidino group present on arginine- and homoarginine-terminal peptides. Modification of the separation conditions employed during LC demonstrates that relative abundances of electrosprayed peptides ions rely mostly on peptide structure.