Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
5	No pellet observed after MV isolation	A different collection or storage method for plasma (different collection tubes, centrifugation times, among others) was used	If plasma is obtained from a third party, make sure the isolation method for plasma is consistent with Steps 1 and 2
		An insufficient amount of plasma (<1 mL) was initially used	Make sure to use at least 1 mL of plasma
		Isolation of plasma after blood draw took too much time, causing loss/degradation of the sample	Be sure to isolate the plasma promptly after blood draw
		Plasma was frozen and then was not thawed according to the instructions above, causing loss/degradation of the sample	Follow the instructions given for thawing of plasma
13	Highly viscous and/or cloudy samples are obtained after lysis	Some patients’ plasma is more viscous than others. This can be associated with different diseases	A certain level of cloudiness is normal. Sonicate the samples to reduce viscosity before proceeding to the next step and/or increase the EV lysis volume
		Samples from different patients were pooled	Sonicate the samples to reduce viscosity before proceeding to the next step and/or increase the EV lysis volume
		The samples contain a high protein content	Sonicate the samples to reduce viscosity before proceeding to the next step and/or increase the EV lysis volume
39	A yellowish sample was obtained after quenching with sodium sulfite	The solution was not quenched completely or the sample contains a low amount of glycopeptides	Add an extra 2-3 μL of 1 M sodium sulfite and incubate for 10 min with shaking in the dark
49	The StageTips clog	If a StageTip flow is slow or stops during glycopeptide desalting, it usually is due to remaining hydrazide beads or any other material in the solution	Make sure to avoid taking any remaining material, especially beads, while transferring the eluates from the hydrazide beads. You can also increase the spin time by a couple of minutes and set the centrifugation speed 200g higher
		StageTip clogging can also be due to excessive force used to press the material into the tip.	Make sure to change packed tip and that the material in the tip is not pressed too tightly.
53	Plasma proteins are highly abundant	This usually happens when not all supernatant is removed at the time of EV isolation (Steps 3-11).	The MV pellet is usually solid enough to pipette everything out without the pellet being disturbed. The same is true for the exosome pellet, even if this pellet is not visible (most of the time). Make sure to remove all of the supernatant
	The yield of glycopeptides and phosphopeptides is low	Some common issues that lead to poor recovery are inefficient proteome digestion, use of old buffers, and sample contamination during the enrichment steps	If the yield is lower than expected, several parameters should be revised in the data search, including the digestion parameters, and phosphopeptide and glycopeptide enrichment specificity (selectivity). If large numbers of non-phosphorylated or non-glycosylated peptides are observed, it is most likely due to inefficient washing of the beads