Chapter 4 : Protein Analysis in the Clinical Immunology Laboratory

Agarose gel electrophoresis separation of plasma proteins based on their electrophoretic mobility. α1Ac, α1-antichymotrypsin; α1Ag, α1-acid glycoprotein; Alb, albumin, α1At, α1-antitrypsin; AT3, antithrombin III; C1Inh, C1 esterase inhibitor; Cer, ceruloplasmin; CRP, C-reactive protein; FB, factor B; Fibr, fibrinogen; Gc, Gc-globulin (vitamin D-binding protein); Hpt, haptoglobin; Hpx, hemopexin; α-Lp, α-lipoprotein; β-Lp, β-lipoprotein; α2-M, α1-macroglobulin; Pl, plasminogen; Pre A, prealbumin; Tf, transferrin. Reproduced from reference 11 with permission from Elsevier Ltd.

Representation of experimental formats for antibody microarrays. (I) Direct labeling of proteins and detection using a single antibody captured on the microarray. (II) Indirect assessment using a two-antibody system. The capture and the first detection antibody are matched, and the detector antibody is measured using a labeled second (readout) antibody.

Representation of antigen microarray. Antigens are “caught” on the microarray through many different processes, which can be generalized as “printing.” Antibodies against specific antigen are detected using a labeled second detector antibody. TTG, tissue transglutaminase.

OPA for detection of functional antipneumococcal antibodies in serum after vaccination. The use of fluorescently labeled pneumococcal polysaccharide-coated beads permits flow cytometric assessment of the opsonic capability of antipneumococcal antibodies in serum, in the presence of exogenous complement, by measuring phagocytic uptake using a differentiated granulocyte cell line.

(A) Fluorescent microscopy showing differentiated granulocyte cell line with phagocytosed (opsonized) fluorescent beads coated with pneumococcal polysaccharide. (B) Flow cytometric analysis of fluorescent signal from phagocytosed beads. Each bead is coated with a unique polysaccharide, and a multiplex mixture of beads is used to measure functional antibodies produced for each specific pneumococcal serotype. (C) The reciprocal dilution that demonstrates 50% maximal uptake of labeled beads is calculated as the phagocytic titer.

(A) Flow cytometric expression of CD64 on “resting” neutrophils (left panel), which is essentially absent because it is not a constitutive marker in this cellular subset. CD64 expression on neutrophils from a patient with bacterial sepsis (blue peak, right panel), as it is a marker expressed on neutrophil activation specifically in the context of infectious stimuli. (B) Flow cytometric analysis of an intracellular protein, Btk, in B cells from a healthy donor (left panel) and in monocytes (Monos) from a healthy donor (middle panel), and the absence of Btk protein in monocytes from a patient with XLA (right panel). Red peak, isotype control; blue peak, specific anti-human Btk antibody. (C) Flow cytometric analysis of modification (e.g., phosphorylation) of intracellular cell signaling proteins. Alterations in function of cell signaling pathways can be assessed after cell stimulation and activation. The assay format can be singleplex (stimulus 1 or stimulus 2 only) or multiplex (stimulus 1 and stimulus 2 in combination), assessing different cell signaling proteins simultaneously (i.e., single experiment). Panel C reproduced from reference 11 with permission from Elsevier Ltd.

Mass spectrum of a monoclonal immunoglobulin light chain protein showing the multiply charged ions produced by ESI (top). Each peak represents the same light chain protein with a different number of protons attached, which changes each ion's mass/charge ratio (m/z). The molecular mass of the light chain is determined by converting each peak to the uncharged state through an algorithm performed by a computer program (bottom).

Mass spectrum of the same monoclonal immunoglobulin light chain protein shown in Fig. 7 ionized using MALDI. The spectrum clearly shows the prevalence of the +1 and +2 charge states as compared with the highly charged ions created by ESI. The molecular mass of the light chain with the +1 charge state is determined by subtracting the mass of a proton.

A graphical representation of the transmission through a triple-quadrupole mass spectrometer for a specific proteotypic peptide ion quantified using SRM. The intact peptide ion is created by ESI and is represented by the green balls. The peptide ion is selected in Q1 and fragmented in the collision cell (Q2), and then a proteotypic peptide-specific fragment ion is transmitted through Q3 on to the detector.

Diagram of an Orbitrap analyzer showing how ions are injected into the Orbitrap via the C-trap and then allowed to orbit the isolated electrodes. The induction current made by the ions is detected and then converted to m/z using the Fourier transform.

Stepwise depiction of a shotgun LC-MS/MS experiment. The figure shows how a tryptic peptide mixture is first separated by LC, ionized, and then scanned in the mass spectrometer. Tryptic peptides with sufficient signal are automatically selected for fragmentation, and the observed MS/MS data are automatically compared to a protein database. A list is provided of the best matches to tryptic peptides derived from proteins in the database.

Top-down MS of adalimumab spiked into normal serum. The ion at m/z = 1,233 in the top spectrum matches the +19 charge state ion from the κ light chain of adalimumab and was selected for top-down MS. The arrow points to the fragment ion mass spectrum shown below. The labeled fragment ions match the expected masses for fragment ions from the C-terminal portion of the κ light chain, which contains the constant region. The calculated y ion masses for the κ light chain constant region-specific amino acid sequence are shown in the table.