GSAT501: Proteomics Peptide sequencing

GSAT501: Proteomics Peptide sequencing Nichollas Scott

The sum of the parts can be more informative then the whole • How do we get from this? 1916.91455 z=1 100 90 259.09183 z=1 80 70 60 Relative Abundance 50 40 916.50836 z=1 586.35480 30 z=1 1856.79382 366.13855 1288.67346 z=? z=1 845.47046 z=1 20 z=1 1086.61328 1359.70862 z=1 z=1 10 1716.83411 z=1 0 200 400 600 800 1000 1200 1400 1600 1800 m/z • To this this?

Things to keep in mind: Isotopic distributions of typical peptides • Peptides typically only contain Carbon, Nitrogen, Hydrogen Oxygen, monoisotopic mass most abundant for molecules <~2000 Da

Example of typical biomolecule isotope distributions 619.32267 z=2 100 982.71539 z=3 90 100 983.04927 982.38102 90 80 z=3 z=3 619.82402 80 70 z=2 70 60 983.38293 60 Relative Abundance z=3 50 Relative Abundance 50 40 40 620.32532 30 30 z=2 20 20 620.82651 z=2 10 10 0 0 981.5 982.0 982.5 983.0 983.5 984.0 984.5 985.0 617 618 619 620 621 622 623 624 625 m/z m/z

Things to keep in mind: Spectral processing terminology • Deconvolution • Monoisotopic mass +2 +1 % De-isotoping m/z m/z m/z m/z m/z

Peptide sequencing: garbage in-garbage out • Best results are obtained by collecting high quality data then processing/evaluating it in the following way; • Remove/determine chemical or instrument generate noise • Recalibrate/identify mass accuracy to account for drift in accuracy/ instrument changes • Identify ions due to co-isolation • These parameters have all been shown to improve peptide identifications and the combination and approaches used by workflows vary

Chemical and instrumental related peaks • Chemical noise and instrumental artifact are common but m/z patterns are distinct from peptide related ions • Consider the theoretical peptide fragment ion map for m/z 635 – 695 for >100000 spectra. Fragment ions occupy discrete m/z regions EgertsonJD et al J Am Soc Mass Spectrom. (2012) 23(12):2075-82..

Chemical and instrumental related peaks 1231.05 1212.59 100 100 90 90 80 80 70 70 1213.59 1267.98 1239.87 1264.00 60 60 1253.44 1240.76 Relative Abundance Relative Abundance 50 50 1246.06 1268.97 1252.19 1237.32 40 40 1250.75 1255.82 1221.99 1234.23 30 30 1226.07 1214.60 20 20 1212.04 10 10 1215.60 1210.56 1221.55 1219.93 1209.62 1216.60 0 0 • Which spectra shows chemical noise, which is a real fragment ion? 1220 1208 1230 1210 1212 1240 1214 1250 1216 1218 1260 1220 1270 1222 m/z m/z

MS accuracy over time • Instrumentation calibration drifts over time and in response to the environment of the instruments (temperature) Makarov A et al Anal. Chem., 2006, 78 (7), pp 2113–2120

MS accuracy within a single scan • Within a single scan the mass error will be uniform or observed to obey a linear relationship • Each red dot = match by search algorithm MASCOT • Which ones are real?

Recalibration using an internal calibrant • Deviations in mass can be corrected introducing a calibrant during the acquisition Wenger C D et al. Mol Cell Proteomics 2010;9:754-763

Isolation of precursor: co-isolation of multiple ions • ~100000 ions are detected within a single LC run and due to this complexity it is common for two or more precursors to be co-isolated at the same time Houel S, et al J Proteome Res. 2010 6; 9(8): 4152–4160

Isolation of precursor: co-isolation of multiple ions • Resulting spectra is known as a Chimeric spectra, ~30-50% of MS/MS can show evidence of co-isolation No match when using standard database searching Houel S, et al J Proteome Res. 2010 6; 9(8): 4152–4160

Isolation of precursors: Example 744.84 969.49 1279.92 969.99 z=2 z=2 z=3 100 100 100 z=2 745.34 z=2 90 90 90 • Which example will give a spectra containing predominantly product ions from a single precursor • Isolation window used is 2 m/z wide 80 80 80 1280.59 z=3 70 70 70 60 60 60 1279.59 A. B. C. z=3 Relative Abundance Relative Abundance Relative Abundance 970.49 50 50 50 744.90 745.84 745.40 z=2 z=? z=2 z=? 1280.92 40 40 40 z=3 30 30 30 970.61 970.43 z=5 z=2 745.90 970.94 746.35 z=? z=2 20 20 20 z=2 971.43 1281.26 z=2 z=3 10 10 971.62 10 1281.59 z=5 z=3 0 0 0 968.0 1276 968.5 1277 969.0 1278 969.5 1279 970.0 1280 970.5 1281 971.0 1282 971.5 972.0 1283 972.5 1284 973.0 744.0 744.5 745.0 745.5 746.0 746.5 747.0 747.5 m/z m/z m/z

MS of non-peptide ions 89.0519 3600 3400 133.0767 3200 • Typically dominated by low mass ions, • Example: Polyethylene glycol 3000 2800 2600 2400 177.1034 2200 2000 Intensity, counts 1800 1600 1400 1200 1000 800 459.2697 600 221.1303 400 265.1580 200 415.2459 371.2197 239.1405 117.0852 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z, amu

Peptide sequencing: Example • Which ions are most likely peptide related fragments

Peptide sequencing: Example

Peptide sequencing: Example 291 =QY or YQ 890.5+163.1=1053.6 890.5+128.1=1018.6 1018.6-17(NH2)

Peptide sequencing: Example b1 are rarely observed

Guidelines for confident peptide sequencing • Only y-, b-, or a-ions or associated peaks arising due to water or amine loss are considered • At least 5 isotopically resolved, independent fragment peaks must match theoretical peptide fragments. • 2. All isotopically resolved higher mass than that of the doubly charged parent mass must match theoretical peptide fragments. • 3. All isotopically resolved peaks with intensities higher than 20% of the maximum intensity must match theoretical peptide fragments. • 4. The difference in the mass errors of neighboring fragment peaks that are within 200 Da of each other must be low (threshold instrument dependent). Chen Y et al J. Proteome Res., 2005, 4 (3), pp 998–1005

Conclusion • Understanding the expected properties of peptides aid in the determination of the sequence • Multiple levels of processing are typically done to MS data to generate peptide identifications • Guidelines exist to assess the assigned peptide sequence

GSAT501: Proteomics Peptide sequencing