Detection of bound and free IGF-1 and IGF-2 in human plasma via biomolecular interaction analysis mass spectrometry

AbstractInsulin like growth factor (IGF)-1 and IGF-2 were assayed from human plasma via biomolecular interaction analysis mass spectrometry, utilizing antibodies as ligands for affinity retrieval. Detection of both targeted and non-targeted IGFs in the mass spectra indicated possible protein complex retrieval by the individual antibodies. A series of control experiments eliminated the possibility of analyte cross-walking between flow cells, significant antibodies cross-reactivity, and direct IGF interactions. To disrupt the putative protein complex and release its constituent proteins, plasma samples were treated with detergents. An SDS-treated plasma yielded IGF signals in a different ratio than the one observed in the mass spectra from the non-treated plasma, suggesting disruption of the protein complex, and its retrieval from non-treated plasma. Novel truncated IGF-2 variant, missing its N-terminal Alanine, was detected in all mass spectra

Mass Spectrometry Of Nicotinic Acetylcholine Receptors And Associated Proteins As Models For Complex Transmembrane Proteins

Studies were conducted to optimize matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI TOF MS) in analyzing the composition of nicotinic acetylcholine receptors (nAChR) from Torpedo californica electric tissue in their membrane-bound, detergent-solubilized, and affinity-purified states. Mass spectra obtained from nAChR-rich membrane fractions gave reasonably good representations of protein compositions indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of those same samples. Efficiency of extraction of nAChR from membranes was not markedly different for most detergents, but quality and signal size of mass spectra were clearly influenced by detergent composition and concentration, protein concentration, and MALDI matrix composition. The best spectra, allowing detection and accurate size determinations for samples containing as little as 10 fmol of pure nAChR, were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix. Although informative spectra could be obtained for nAChR affinity purified on α-cobratoxin (Naja naja siamensis) columns and extracted using sinapinic acid, superior spectra with much higher signal:noise were obtained if extraction media contained Triton X-100 or sodium dodecyl sulfate. nAChR subunit masses determined were similar regardless of the membrane-associated, detergent-solubilized, or affinity-purified state of the preparation. These studies illustrate how masses can be determined for nAChR subunits and for other protein components in Torpedo membrane preparations, such as RAPsyn and Na+-K+-ATPase α and β subunits. They also provide an underpinning for streamlined analysis of the composition of complex transmembrane proteins using MALDI TOF MS. © 2002 Elsevier Science (USA)

Targeted selected reaction monitoring mass spectrometric immunoassay for insulin-like growth factor 1.

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories