Isoforms of wild type proteins often appear as low molecular weight bands on SDS‐PAGE

Abstract

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE), is a technique commonly used to detect specific proteins. SDS-PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography - mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non-malignant HEK293 and cancerous MDA-MB231 (MB231) cells separated using SDS-PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS-PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer-therapy studies.

About three‐fourths of mouse proteins unexpectedly appear at a low position of SDS‐PAGE, often as additional isoforms, questioning whether all protein isoforms have been eliminated in gene‐knockout cells or organisms

“…For example, five heat shock proteins (Hspa1a, Hspa8, Hspd1, Hsp90aa1, and Hsp90ab1) have their identified peptides evenly distributed across the entire protein with a high CR. In contrast, for more than 80% of these 40 genes, their proteins have one or more large regions lacking a detected peptide, also similar to what we have reported previously . It is worth mentioning that many lengthy proteins have repeated sequences, such as the Plec1 (Figure , lower panel), usually to reinforce some important functional domains.…”

“…Since it is unlikely that a large region of a protein sequence lacks a trypsin‐digested peptide for LC–MS/MS detection, as shown in Figures , and , a likely reason for the detection of the smaller group, generally speaking, is that the detected protein is either a degraded fraction or a smaller isoform lacking part(s) of the sequence. The LC–MS/MS results provide coverage rate (CR) of identified AAs (Table S2, column D), that is, the total AAs of all identified peptides as the percentage of the total AAs of the largest protein isoform listed in the NCBI database, as illustrated in Figure a using the 6PGL protein as an example . A larger CR indicates that the detected protein isoform is closer to the full‐length form, with a CR of 1 (100%), which will not occur in any real case, indicating that all AAs are LC–MS/MS identified.…”

“…Considering that the prestained protein markers may not be accurate enough and protein migration in SDS-PAGE can be affected by different factors, as described before, 25,26 we arbitrarily allow those proteins with a TMM in the range of 23-29 kD, which is about 10% larger or smaller than 26-kD, to be considered the expected gene products, herein referred to as "the WT," as described before. 25,26 Those proteins with their TMMs above this WT range are categorized into a "smaller-group," while those with their TMMs below this WT range are grouped into a "larger-group," because their positions in the gel were smaller or larger, respectively, than their WT TMMs ( Table 2). Because many genes are expressed as multiple protein isoforms, the categorization is made based on the largest isoform listed in the NCBI database, which may or may not be the canonical WT.…”

“…We have actually mapped the LC-MS/MS-detected peptides onto the largest isoform from 40 genes with the largest TMM among all genes detected and found that only a few of them have the identified peptides evenly distributed throughout the entire sequence (data not shown), similar to what we have reported previously. 25,26 For example, five heat shock proteins (Hspa1a, Hspa8, Hspd1, Hsp90aa1, and Hsp90ab1) have their identified peptides evenly distributed across the entire protein with a high CR. In contrast, for more than 80% of these 40 genes, their proteins have one or more large regions lacking a detected peptide, also similar to what we have reported previously.…”

Identification of a protein associated with the activity of cytokine‑induced killer cells

“…In the present study, it was demonstrated that the CIK activity was significant inhibited by gentamicin, and further research revealed that a protein P43 was associated with CIK activity. To determine the identity of the P43 protein, 1-DE with LC-MS/MC was performed (24,25). The P43 protein was successfully identified as human haptoglobin.…”