Probably less than one-tenth of the genes produce only the wild type protein without at least one additional protein isoform in some human cancer cell lines

Abstract

To estimate how many genes produce multiple protein isoforms, we electrophoresed proteins from MCF7 and MDA-MB231 (MB231) human breast cancer cells in SDS-PAGE and excised narrow stripes of the gel at the 48kD, 55kD and 72kD. Proteins in these stripes were identified using liquid chromatography and tandem mass spectrometry. A total of 765, 750 and 679 proteins from MB231 cells, as well as 470, 390 and 490 proteins from MCF7 cells, were identified from the 48kD, 55kD and 72kD stripes, respectively. We arbitrarily allowed a 10% technical variation from the proteins’ theoretical molecular mass (TMM) and considered those proteins with their TMMs within the 43-53 kD, 49-61 kD and 65-79 kD ranges as the wild type (WT) expected from the corresponding stripe, whereas those with a TMM above or below this range as a smaller- or larger-group, respectively. Only 263 (34.4%), 269 (35.9%) and 151 (22.2%) proteins from MB231 cells and 117 (24.9%), 135 (34.6%) and 130 (26.5%) proteins from MCF7 cells from the 48kD, 55kD and 72kD stripes, respectively, belonged to the WT, while the remaining majority belonged to the smaller- or larger-groups. Only about 3-16%, on average about 10% regardless of the stripe and cell line, of the proteins appeared in only one stripe and within the WT range, while the remaining preponderance appeared also in additional stripe(s) or had a larger or smaller TMM. We conclude that few (fewer than 10%) of the human genes produce only the WT protein without additional isoform(s).

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

“…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.…”

“…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. 25,26 It is worth mentioning that many lengthy proteins have repeated sequences, such as the Plec1 (Figure 4, lower panel), usually to reinforce some important functional domains. It is a plausible conjecture that the smaller isoforms would be the ones lacking one or more of the repeated regions.…”

ACTB and GAPDH proteins appear at multiple positions of SDS-PAGE and may not be suitable for serving as reference genes for the protein level determination in such techniques as Western blotting

“…This study contained two separate experiments of SDS-PAGE and LC-MS/MS, as described previously [22,23]. In the first experiment [22], human breast cancer cell line MDA-MB231 (MB231) and human embryonic kidney cell line HEK293 were cultured as routine at 37 C in an incubator with 5% CO 2 in 10-cm dishes with a DMEM medium containing 10% bovine fetal serum.…”

“…Our studies with this oversimplified top-down approach surprisingly show that most proteins identified are not supposed to appear at a given position of SDS-PAGE because their theoretical molecular masses are either much too large or much too small. Since Western blotting (WB) in most, if not all, cases can detect proteins at the expected position of SDS-PAGE, we concluded that most of those proteins detected unexpectedly at a given SDS-PAGE position might be additional isoforms besides the canonical or the wild type (WT) form [22][23][24]. For instance, ACTB and GAPDH are proteins of about 41.7 kD and 36 kD, respectively, and they can indeed be detected at the corresponding position of SDS-PAGE using WB in probably all published studies.…”