LMD/MS analysis of a case of AL-kappa amyloidosis (case 1). (A) Bone marrow specimen with interstitial nodules of amyloid deposition (Case 1). (Left) CR-stained section viewed under bright field confirming congophilia of the amyloid deposits. (Middle) CR-stained section viewed under fluorescent light source. The congophilic deposits give bright red fluorescence, confirming amyloid deposition. The area selected for microdissection is circled with a red line. (Right) Same area selected in the middle panel after microdissection of the amyloid plaque. Photograph (left) was taken with a DP70 Olympus camera (Olympus) with an Olympus BX51 microscope (Olympus); images were acquired by the use of a DP Controller 2002 (Olympus) and processed with Adobe Photoshop Version 7.0 (Adobe Systems). Other photographs (middle and right) were taken with a HV-D20 Hitachi camera (Hitachi Kokusia Electric Inc) by use of a Leica DM6000B microscope (Leica Microsystems GmbH); images were acquired with Leica Laser Microdissection LMD software (Version 6.6.0; Leica Microsystems). Fluorescence images were obtained by use of a triple band pass filter (B/G/R fluorescence filter; Leica Microsystems). Original magnifications: ×100 (left); ×200 (middle and right). (B) The list of proteins identified from the microdissected amyloid fragments are shown above in panel A. The proteins are listed according to the abundance they were represented in the sample. The panel shows the protein accession code in the UniProt database,¹⁹  the molecular weight of the protein (MW), the results of the blank control sample, and 4 different microdissections (1-4) For each protein identified in each sample, statistical probability is indicated as a percentage. The numbers in parentheses indicate the number of unique peptides identified belonging to a given protein. Of the 4 common types of systemic amyloidosis specifically studied (SAA, TTR, IGK, IGL), the samples contained peptides only belonging to IGK constant region (Accession: KAC_HUMAN, highlighted in yellow with red text). In addition, the samples contained several proteins known to be associated with amyloid deposits, such as apolipoprotein E (Accession: APOE_HUMAN), apolipoprotein A4 (Accession: APOA4_HUMAN), SAP (Accession: SAMP_HUMAN), and apolipoprotein A1 (Accession: APOA1_HUMAN). These are indicated in blue text. Other proteins seen include normal components of bone marrow stroma such as vitronectin (Accession: VTNC_HUMAN) and collagen (Accession: CO6A3_HUMAN). Given that the only amyloidogenic protein belonging to the 4 common types of amyloidosis was IGK, this case was typed AL-kappa-type by MS. This result was consistent with the previous gold standard diagnosis. (C) Detailed results of IGK constant region (Accession: IGKC_HUMAN) identified in all 4 cases samples. Probability of protein identification, the number of unique peptides, unique spectra, and total spectra and percent coverage of the protein are shown. A more detailed analysis of the proteomic data is provided in supplemental Figure 1.