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Protein biomarker discovery and validation are crucial for diagnosis, prognosis, and theranostics of human pathologies; 'omics' approaches bring new insights in this field. In particular, the combination of immuno-sensors in array format with mass spectrometry efficiently extends the classical immunoassay format and includes molecular characterization. Here, we coupled surface plasmon resonance imaging (SPRi) with MALDI-TOF mass spectrometry in a hyphenated technique which enables multiplexed quantification of binding by SPRi and molecular characterization of interacting partners by subsequent MS analysis. This adds specificity, because MS enables differentiation of molecules that are difficult to distinguish by use of antibodies, for example truncation variants or protein isoforms. Proof of concept was established for detection, identification, and characterization of a potential breast cancer marker, the LAG3 protein, at ~1 μg mL, added to human plasma. The analytical performance of this new method, dubbed 'SUPRA-MS', was established, particularly its specificity (S/N > 10) and reliability (100 % LAG3 identification with high significant mascot score >87.9). The adjusted format for rapid, collective, and automated on-chip MALDI-MS analysis is robust at the femtomole level and has numerous potential applications in proteomics. [ABSTRACT FROM AUTHOR]