Daily Archives: May 22, 2023

Peaks per hexamer of SP-D

Peaks per hexamer were counted three ways –
1. IMAGE = my counts of bright spots in the AFM image (aka peaks). This was recorded for each trimer,  hexamer, and collated for each dodecamer (N=14), and for each image processing filter and for each signal processing function.

2. PLOTS = my counts from the “image” of each of the plots created by ImageJ from my trace through the center of each hexamer in the direction of the CRD peak to the opposite CRD (as in, end to end). Directions of the segmented line through each hexamer were ALWAYS traced in the same direction (left to right) for all the peak finding and peak counting apps.

3. SIGNAL = peak counts were generated from 5 approaches (Python/Scipy app, Stack  Overflow app, Octave (two functions; ipeakM80, AFPPxy), a PeakValleyDetectionTemplate.xlsx) each using using the same grayscale .csv files created from traces in ImageJ.

My peak detection from the actual image consistently consistently fell between counts from the plots themselves, and the peak count generated by signal processing functions.  Mean peak counts from three methods continues to identify 15 peaks per hexamer.

Summary table below shows both the individual values (896 trimer counts and all processing types), and individual dodecamer counts (N=14, X+/SD).  (image=my counts from each image) vs plot  (=my counts from each plot from each image recorded by ImageJ). These two counts are not significant at p < .05. However, there is a significant difference between my peak counts from the ImageJ plot and the peak counts that is tallied from the signal processing functions. ( p-value is .0119); There is no significant difference in the number of peaks found when I count peaks directly from the image vs the number of peaks found with signal processing. Results with an N of individual trimer counts (N=896), and the mean and SD from counts from each dodecamer (N=14).

data for 12 dodecamers is here.

and comments from a previous post here.

The graphic above separates the peak finding into separate categories (highlighting the vast majority of the counts were from signal processing functions). It shows total peaks counted from the image itself (image ONLY), and my counts of peak number from the plots from those images (plot ONLY), the peak counts after all signal processing functions (none of my counts)(signal ONLY).  The bottom row is all counts all methods, all the time (EVERYTHING).  LIttle variation, basically the same number as found a year or two ago. 15 peaks per hexamer

Comparing 4 sets of peak finding for SP-D

Four sets of data are below (gathered incrememtally – from 6 to 14 dodecamers) were examined for number of peaks, and sub-peaks per trimer.   Each dataset includes the molecules from the prior set, i.e. the same initial 6 are part of the new 14 dodecamer data. An image of one of those 14 dodecamers analyzed is shown below with color-matching circles of where the 8 peaks per trimer are align on the molecule. You will count 9 dots. 

The initial number of peaks per each hexamer in a dodecamer was found using signal and image processing on many occasions and using over 1000 plots. That number influenced the division of each plot of a hexamer – but ultimately using the plot from the image and the peak detection plots as a resource for that division. The sub-peak of the N term peak  detected in dodecamers was detected less than 1% of the time (very pale green), (but may be more prominent in multimers), and the peak called “tiny peak” (purple)  on the downslope of each side of the N term center peak was detected about 33% of the time. These were data were included when they appeared.

At opposite ends of the hexamer the CRD peak (dark orange) and neck peak (yellow) occur.  The neck peak is sometimes concealed by the overlap of the CRD peak(s) (which in seem to be a flexible part of a largely rigid molecule), and can lie during preparation in a floppy cluster obscuring a nearby neck peak.  The neck peak is detected as a unique peak about 44% of the time.

Of the “not yet reported peaks” there is the tiny peak (purple) between the N term peak and the glycosylation peak, and the three peaks just lateral to the glycosylation peak. The latter three peaks are as follows: one large peak (detected almost 100% of the time) which is about the same size as the glycosylation peak, and two smaller peaks (pink and white – matching the color of the rows of data). Circles are approximate representation of relative peak widths.

This leaves three additional, as yet NOT reported peaks, bringing the total number of peaks not yet reported to 5.  The percent detection is given below in progressive sets of data.

The number of peaks (top row, number of peaks, number of trimers, and subpeaks (from 1 peak to 8 subpeaks, in columns)  in each dataset is shown below (color markers for peaks remains consistent throughout (and also on previous and newer posts).  The glycosylation peak (light green row of data) and the adjacent as yet unreported peak (darker green row of data) show consistent, multiple sub-peaks. These sub-peaks are found mainly by the signal processing functions. Addition of dodecamers to the initial dataset show little change.