Immunoprecipitation and Mass Spectrometry Analysis of Amyloid-Beta Peptides

Immunoprecipitation and mass spectrometry (IP-MS) of Aβ and Aβ-like peptides in cell-free assay or conditioned media were performed as previously described [7 (link), 15 (link), 24 (link), 28 (link)–30 (link)]. Briefly, the peptides were immunoprecipitated using anti Aβ Ab5 antibody bound to sheep anti-mouse IgG magnetic Dynabeads (Life Technologies) and eluted with 0.1% trifluoroacetic acid (TFA) in water. COOH-terminal fragments (CTFs) were immunoprecipitated with anti-FLAG M2 magnetic beads (Sigma). Eluted samples were mixed 2:1 with saturated α-cyano-4-hydroxycinnamic acid (CHCA) matrix (Sigma) in a mixture of acetonitrile (60%) and methanol (40%) and loaded onto a CHCA pretreated MSP 96 grounded steel target (Bruker, Billerica, MA). Spectra were collected and processed with a Bruker Microflex LRF-MALDI-TOF mass spectrometer using the flexControl and flexAnalysis software.

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Lessard C.B., Rodriguez E., Ladd T.B., Minter L.M., Osborne B.A., Miele L., Golde T.E, & Ran Y. (2020). γ-Secretase modulators exhibit selectivity for modulation of APP cleavage but inverse γ-secretase modulators do not. Alzheimer's Research & Therapy, 12, 61.

Publication 2020

sheep anti-mouse IgG magnetic Dynabeads anti-FLAG M2 magnetic beads α-cyano-4-hydroxycinnamic acid (CHCA) matrix Microflex LRF-MALDI-TOF mass spectrometer

Acetonitrile Anti igg Antibody Assay Cell Conditioned media Hydroxycinnamic acid Immunoprecipitation Maldi Mass spectrometry Methanol Mouse Peptides Sheep Steel Trifluoroacetic acid

Corresponding Organization :

Other organizations : University of Florida, University of Massachusetts Amherst, Louisiana State University Health Sciences Center New Orleans

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Variable analysis

independent variables

Immunoprecipitation of Aβ and Aβ-like peptides

dependent variables

Mass spectrometry (MS) analysis of the immunoprecipitated Aβ and Aβ-like peptides

control variables

The immunoprecipitation and MS analysis were performed as previously described in the referenced studies [7, 15, 24, 28-30]

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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