Label-free mass spectrometry exploits dozens of detected peptides to quantify lamins in wildtype and knockdown cells

Abstract

L abel-free quantitation and characterization of proteins by mass spectrometry (ms) is now feasible, especially for moderately expressed structural proteins such as lamins that typically yield dozens of tryptic peptides from tissue cells. using standard cell culture samples, we describe general algorithms for quantitative analysis of peptides identified in liquid chromatography tandem mass spectrometry (Lc-ms/ms). the algorithms were foundational to the discovery that the absolute stoichiometry of a-type to b-type lamins scales with tissue stiffness (swift et al., Science 2013). isoform dominance helps make sense of why mutations and changes with age of mechanosensitive lamin-a,c only affect "stiff" tissues such as heart, muscle, bone, or even fat, but not brain. a peak ratio fingerprinting (prf) algorithm is elaborated here through its application to lamin-a,c knockdown. after demonstrating the large dynamic range of prf using calibrated mixtures of human and mouse lysates, we validate measurements of partial knockdown with standard cell biology analyses using quantitative immunofluorescence and immunoblotting. optimal sets of ms-detected peptides as determined by prf demonstrate that the strongest peptide signals are not necessarily the most reliable for quantitation. after lamin-a,c knockdown, prf computes an invariant set of "housekeeping" proteins as part of a broader proteomic analysis that also shows the proteome of mesenchymal stem cells (mscs) is more broadly perturbed than that of a human epithelial cancer line (a549s), with particular variation in nuclear and cytoskeletal proteins. these methods offer exciting prospects for basic and clinical studies of lamin-a,c as well as other ms-detectable proteins.

Do lamin A and lamin C have unique roles?

“…Noteworthy, the nucleoplasmic fraction of lamin A but not lamin C had increased mobility in cells with decreased amount of nucleoplasmic lamin B1 (Shimi et al 2008). Very recently, a study by Swift has shown that siRNA was able to knockdown more lamin A than lamin C (Swift et al 2013a). These findings might indicate that lamin A is less stable and more susceptible to various influences than lamin C. Another recent report by Swift et al has demonstrated increased lamin A expression during differentiation of bone (Swift et al 2013b).…”

Matrix Elasticity Regulates Lamin-A,C Phosphorylation and Turnover with Feedback to Actomyosin

“…Quantification of proteins by label-free MS is described in the Supplemental Information and earlier work [34]. MS response to phosphorylation at S22 and S390 was calibrated using synthetic versions of tryptic peptides (SGAQASSTPLSPTR, SGAQASSTPL[pSer22]PTR, LRLSPSPTSQR LRL[pSer390]PSPTSQR; GenScript), which were spiked into a tryptic cell digests (60 – 80 kDa MW band) (Fig.…”

The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue

“…1B, left panel). By using quantitative label-free mass spectrometry for proteomic profiling (Swift et al, 2013a), we have shown that collagens and other ECM-associated proteins scale with tissue elasticity (Swift et al, 2013b). Mass spectrometry was also used to quantify ,100 of the most abundant proteins in the cytoskeleton and nucleus, and we found that the elasticity of bulk tissue is strongly correlated with the composition of the nuclear lamina in terms of its content of Atype and B-type lamins (Fig.…”