FAQs

Immuno-affinity-mass spectrometry combines a specific enrichment step (typically using an anti-peptide antibody) to capture a pre-selected target peptide followed by quantitative readout using mass spec. This approach combines the main advantages of immunoassays (sensitivity and throughput) with those of mass spectrometry (absolute structural specificity, true internal standards, and wide dynamic range).

SISCAPA is often used where immunoassays run into problems such as interferences, inability to distinguish similar molecular forms, solubility issues (e.g., for membrane proteins) and multiplexing a set of assays into a panel.

Or, why enrich a peptide instead of a protein? By digesting the sample proteins first, the SISCAPA workflow removes a whole host of assay interferences due to protein:protein interactions, and then selects a carefully chosen surrogate peptide for measurement. Enrichment at the protein level, prior to digestion, leaves the assay subject to protein: protein interferences, makes it difficult to use labeled internal standards, and presents a much more complex sample to the mass spec.

Yes – protocols are available for Agilent Bravo, Beckman Biomek, Hamilton Nimbus and select Tecan lab robots. We recommend use of automated protocols to obtain maximum precision and throughput for SISCAPA assays.

The target peptide should be unique to the protein being measured, perform well in mass spec (i.e., “fly well”), display reproducible digestion kinetics, and induce a good antibody response (e.g., in rabbits), SAT has an established methodology for peptide selection, and extensive experience optimizing all aspects of assay development.

We have panels measuring up to 50 assays at once.

By removing the vast majority of sample matrix peptides, SISCAPA simplifies the sample to a point requiring much less LC separation than the unfractionated sample, and thus less separation time. In some cases SISCAPA samples can be injected into the MS without any LC separation (e.g., by MALDI, RapidFire or Echo).

Yes. By using enough of one antibody to capture all of the corresponding low abundance peptide but using much less of the antibody corresponding to a high abundance peptide) (and thus capturing only a small fraction of what is in the digest), both the peptides are delivered to the MS at more nearly equal abundance, facilitating accurate quantitation. Since the quantity of the target peptide is measured by comparison with a labeled peptide internal standard captured along with the target peptide, the measured target abundance is not affected by this “stoichiometric flattening” method. Peptides differing by abundance by 1,000,000-fold in the digest can thus be presented to the MS with abundances differing less than 1,000-fold.

Compared to an immunoassay, which uses antibody recognition of complex epitope shapes for specificity, SISAPA relies on two-stage mass spectrometry to recognize, and measure, peptides based on sequence. SISCAPA adds to this the specificity of a stringently-selected anti-peptide antibody to yield effectively absolute sequence-based assays specificity.

SISCAPA can be carried out manually, but this can decrease sensitivity and precision due to difficulty of ensuring identical timing of the steps in the processing of all samples. We therefore highly recommend using an automated implementation when possible.

SISCAPA assays typically employ triple-quadrupole MS systems in multiple-reaction monitoring (MRM) mode. However, successful assays have been implemented using MALDI-TOF, QTOF and Orbitrap instruments as well. Any MS capable of measuring the ratio of the sample-derived target peptide and its cognate labeled internal standard can in principle be used.

Yes. We know of at least three multinational CROs successfully using SISCAPA for regulated studies.

Starting from initial peptide selection, it typically takes 7 to 8 months to develop and characterize a SISCAPA assays based on monoclonal anti-peptide antibodies. Creation and selection of these antibodies accounts for the bulk of this time.

SAT’s catalog SISCAPA antibodies were primarily chosen to target human blood proteins of known clinical significance. Current the menu includes more than half of the proteins measured on the widely-used clinical immunoassay platforms, and our goal is to replicate in SISCAPA the entire clinical protein menu of >120 proteins.

Yes. In fact SISCAPA assays developed to measure biomarker proteins in plasma or serum also work well in whole blood, CSF, urine, etc. This is because SISCAPA eliminates almost all the matrix contribution to the final MS peptide sample, and thereby overcomes differences between different sample matrices.

Yes. We have extensive experience using SISCAPA to measure blood proteins in dried blood spots (DBS), and have developed methods for normalizing variations in blood volume and hematocrit that have previously limited the precision of DBS measurements. The high precision of SISCAPA measurements has proven to be critical in tracking small changes in blood biomarkers in longitudinal sample sets, where each subject is used as his/her own control.