Sari tearfully admiting to Ratchet that she tried to use her key to sabotage the Autobot's ship so they couldn't leave and how it wouldn't let them. Especially sad given how it's shown that they're the only real friends she's ever had. It becomes a heartwarming moment when Ratchet admits he's grown attached to her and he'll miss her when they eventually leave.
Sari's breakdown in the episode "Return Of The Headmaster" after finding out that she has no home and doesn't legally exist, and then Bumblebee and Bulkhead comforting her.
When Prowl breaks the nest of bird's eggs in "A Fistful of Energon". Oh, it just breaks your heart to see the look on his face. You're ready to burst into tears. Broken bird's eggs! Oh, it's so sad, go on and admit it...
Omega Supreme's line to Ratchet when he's slowly losing the battle against Starscream's control.
"Shut me down. I want to sleep again."
And the first time he shuts down, with Ratchet reassuring him that he's still a good person.
Everything about Omega Supreme. Everything.
Prowl's Heroic Sacrifice in Endgame Part 2. Try to watch his "I'm sorry, Jazz, I have to do this" face before he lets himself be absorbed by the Allspark and not at least feel sad. Made even worse by Jazz calling to him that they'll find another way as well as his final act to save Optimus.
What makes his smile so effective is how he does it. Pretty much every episode before has him either pull off a simple, normal smile (which is rare) and a smug grin. Cue the last episode when he dies and pulls that particular smile which he has never done before. It culminates all the Character Development he got (the selfish, stoic loner to a willing soldier and loyal friend) and works perfectly for his Heroic Sacrifice.
Watch and listen when everybody gathers after the Lugnut Supreme exploded, and Jazz arrives with Prowl's dead shell. Optimus is the first to move towards Prowl while uttering a single, quiet, disbelieving "No".
Megatron shoots at Optimus when he reaches for Prowl's body. The Decepticon should have been killed by the explosion, but instead, Prowl had to sacrifice himself so the same explosion would not obliberate Detroit and everyone in close vicinity. Prime's quick, vicious counter-attack against Megatron shows no heroics, no speeches, he simply smashes him down and refutes his request for a quick death in battle. Optimus just lost one of his closest friends who promised him just a short time ago he would 'always be at his side'. Now Prowl is dead, it's all because of Megatron, and Optimus has no interest in his grief and rage to be 'merciful'.
Optimus, Elita, and Sentinel's interactions before it all went downhill, the way they had each otherís backs while fending off the spiders, and Elita being left to the spiders with Optimus and Sentinel believing her to be dead is quite sad.
Blackarachnia in the episode definitely needed some hugs too. It must have been unbearably painful for her to live for such a long time in such a disoriented state, and you can't really blame her for switching alliances and not trusting Optimus. And by the end of "Along Came a Spider" she looks off into the moon, crying alone.
Later, Sentinel discovers the truth, and doesn't take it well at all.
Blackarachnia: So that's it?! You just slag your old friend Elita-1?
Ratchet in "The Thrill of the Hunt" summing up his war experiences:
"It's not that I don't want to remember. I have to remember... for those who can't."
When Bumblebee rushes to help an out-of-control teenage Sari, while she screams for him to stay away... and accidentally stabs him. The looks of disbelief and anguish on both of their faces are just heart breaking. Later, Ratchet gets one when he has to stop Sari in a way that almost kills her.
No mention of "Lost and Found"? How about Ratchet's confession to Sari (after she tried to sabotage the ship) that the Autobots had to leave Earth or else the humans, including Sari at the time would be wiped out by the Decepticons. The one that hits the hardest is his saying that he would never forgive himself if that were to happen in a quiet voice.
Blurr's fate. Even if he did survive being crushed into a cube, and even if Cliffjumper is shown carrying the cube around with him in the comic, there's no telling if he'll ever be fixed back into his old shape again.
Wasp wasn't the most likable Autobot, but no one deserves his fate when he's fingered as a spy and sent to the Stockades. When Bulkhead and Bumblebee learn the true identity of the spy, Bulkhead's quiet horror says it best for both of them, having seen what imprisonment did to Wasp.
Bulkhead: (to Bumblebee) You sent an innocent bot to the Stockade.